Sorry that this blog has not been updated in the last two months. I am currently busy, working on my thesis and I have been swamped. I promise that I will have some insightful blog posts when I am done with my final examination.
In the mean time, here are some words from one of my favorite bands, quite out of context for this blog:
"So if you are scared 'cause you think that you are missing out
This is not the ending
No, this is not the ending "
- Tenth Avenue North
Saturday, October 25, 2014
Monday, August 18, 2014
When we left for the brain in a DC shuttle - Toxoplasma gondii
If you know me well, you would know my near-fanatical obsession with the awe-inspiringly beautiful Aurora Borealis. They are especially delightful on cold wintry nights on the icy, wind-swept prairie. And out in the darkness, when one sits, waiting for the beautiful ribbons of light to appear, one may observe the bright, distant balls of gas that are scattered across the sky in a milky band, and sometimes the man-made satellites that look like fast moving stars, seemingly weave in and out of the constellations in the background. If you asked me, I would tell you that astrophysics, sans the formulae, is certainly a field meant for hobby reading. Recently, I enjoyed a documentary series, produced by Discovery Communications, titled "When we left Earth : The NASA Missions", which featured the Mercury, Gemini, Apollo Missions, and NASA's golden age, the Space Shuttle era. The progress in the technology and the ability of humans to go beyond the physical boundaries imposed by the planet and its forces are quite remarkable, and quite interesting to follow, all the more if you were old enough to remember the landing on the moon, [which I am not, but that is not a major deterrent].
Just as the space shuttles were designed to carry payloads and people to the low Earth orbit, the zoonotic apicomplexan, Toxoplasma gondii , uses the dendritic cells of the host to move around uninhabitable spaces and cross barriers that it normally cannot by itself cross. Most comparisons call for the dendritic cells to play the role of a Trojan horse, but the space shuttle analogy works just as well.
Here is a brief overview of the life cycle before we scrutinize Toxoplasma's insane abilities to flag down, hitch rides and hijack shuttles. When the infective bradyzoites (in the cyst) are ingested by the definitive host (domestic cat and other Felids), the sexual cycle ensues in the intestines and oocysts are shed in the feces; quite similar to other Coccidians. However, the asexual cycle in the intermediate hosts is what sets Toxoplasma apart. In humans, sheep, mice and every other imaginable host, infective oocysts differentiate into tachyzoites which invade the intestines, multiply and later migrate in the body to form cysts containing bradyzoites. Favorite spots for cyst formation include the brain, the placenta (resulting in abortion(s)) and the eyes; every one of these organs are "immuno-previleged", that is, they are places where innate immuno-surveillance is lower than in the other organs of the body. Before cysts can be formed, Toxoplasma gondii has to get to these places.The migration of a stage called the tachyzoite is the theme of this post.
Getting to know your shuttle
The Dendritic cells are an enigmatic group of leukocytes, first discovered by Steinman R. in 1973, (Steinman was awarded the 2011 Nobel Prize in Physiology/Medicine, only three days after his death. You know of course, that the Prize is usually not awarded posthumously and this was a rare exception made in good faith by the Nobel Prize Committee, whose members were entirely unaware of the death of the laurate) , that have an absolutely incredible range of activities to perform. These bone marrow derived cells, migrate to their temporary homes and keep themselves entertained by sampling their environment, taking up and processing antigens. They are the great controllers of the adaptive immune system, the gatekeepers, if you will, and live throughout the body, at all possible portals of pathogen entry (the skin, the intestines etc), and the secondary lymph organs. They play key roles in immunological tolerance. Once they encounter an antigen that is deemed dangerous, based on its molecular phenotypic profile, or in simple terms the pattern of its dress, they take it up, process it (read "beat it to a pulp") and later migrate with it to lymph nodes where they "present" the processed antigen (on silver platters called MHC molecules) to T cells that are fit to receive such presents (viz., antigen specific). All the while, the DCs secrete chemokines, through which other cells are activated and the immune response spirals into a cell mediated or humoral response as necessary.
DCs exist as distinct subtypes. The jury is out on whether these subtypes are specialized lineages or just functionally plastic cells. The cellular marker of a mature DC in mouse is the CD11c molecule. Interdigitating DCs carry CD205 as well. CD11b is another marker of interest. In mice spleen, CD4-CD8+, CD4+CD8- and CD4-CD8- DC populations exist. Thymic DCs in mice are predominantly CD4-CD8+CD205+CD11b-. Lymph node DCs are predominantly CD4-CD8-CD205-CD11b+ cells. Langerhans DCs dominate the skin draining LNs, and are CD4-CD8-CD205+CD11b+.
Toxoplasma gondii, the perfect parasite, makes use of the migratory activity of this mysterious cell type. In the intermediate host (the animals on the left side on the picture above; that is, mice, humans and other mammals), the parasite enters the body through the mouth when the infective oocyst stage is accidentally consumed. Oocysts that are shed by a household cat is usually the reason that humans come in contact with the parasite. Toxoplasma oocysts unmask themselves to show their virulent side, becoming fast replicating tachyzoites, by invading the intestinal cells and replicating. The DC in the vicinity leap into action. But, even as they lash out, Toxoplasma tachyzoites invade them and causes them to become hypermotile, even in the absence of chemotactic stimuli from other predilection sites. The precise mechanism by which it uses the DCs as its own personal mode of transportation is only slightly known. Within 2 - 3 hrs the parasite rides its shuttle to the local lymph node, each content to bring the other to the LN.
Leaping to action
The DC response at the first hint of the tachyzoites is the production of IL12, triggered through TLR 11 recognition of a profilin-like molecule of the parasite and CCR5 mediated recognition of cyclophilin 18. Other TLRs that are activated include TLR2 and TLR4, both of which are activated by Toxoplasma's glycosylphosphotidylinositol anchors. Once the TLRs are activated, the response is rapid. IL12 activates NK cells, which secrete the first doses of interferon gamma. By this time, the DCs have migrated to the lymph node and present the Profilin-like molecule on MHC Class II molecules. DCs continue to secrete IL12. Antigen specific CD4+ cells are activated by the cytokine mileu and the presented antigen. They begin to pour out IFN gamma in a perfect example of a positive feedback loop, driving their own differentiation down a Th1 road. And you probably know that Th1 responses are primarily cell mediated. Also, DCs are capable of direct antigen presentation on MHC Class I molecules to CD8+ cells, activating them. Actively dividing tachyzoites are now cleared by the CD4+ and CD8+ cells that have migrated to the infection sites in the intestines and the acute phase of the infection ends. The immune system seems to have done its job.
However, the tachyzoites that infected the DCs that have now reached the local lymph node are alive and well. Once a DC reaches the lymph node, it does not ordinarily leave very easily under normal circumstances. Toxoplasma infected DCs though exhibit a bizarrely hypermotile phenotype that allows them to be steered as Toxoplasma pleases. The CD 11c on the surface is down regulated and the CD 11b is upregulated. The infected cells turn from CD11c+CD11b+/- to CD11c-CD11b+.
According to a study by Lambert et al. (2009), hypermotility of the DCs also depends on the genotype of Toxoplasma, with Type II strains showing "stronger intracellular associations with CD11c+ DCs". Types II and III were better at causing DC movement than Type I, although the latter seemed to have better motility on its own, and was able to cross physiological barriers than the former two.
The destination
In an elegant review article, Galea et al. (2006) clarify the 'immune privileged' status experienced by the brain, which is where Toxoplasma controlled DCs are headed. They reiterate that "immune privilege is relative and not absolute", that is, whilst the brain in its normal condition does not host DCs and other antigen presenting cells, an inflammatory response is an altogether different story. One possible reason for this according to other neuroscientists including Coisne et al. 2006 is the "Differential expression of selectins" and other adhesins like ICAM-1 and VCAM-1.
Barragan et al. in 2005 have shown that when Toxoplasma transmigrates, that is, moves across the blood brain barrier without disrupting it, the layers of intervening cells maintain their structure and integrity. And because this was studied in an electrical organ like the brain, they also measured the Transcellular electrical resistance, which they found to be maintained at a steady state during said parasite-DC migration. Using ICAM-1 antibodies, they discovered that ICAM-1 on the host cell surface was involved and that it interacted with a set of micronemal proteins on Toxoplasma called MIC2.
In a more recent study, Lachenmaier et al. (2011) pinned the exact mechanism of neuroinvasion by stating that CD11b+ cells were the main cell population that were used. They further claimed that both type I and II strains were equally capable of infecting CD11b+/CD11c+ cells and migrating across the blood brain barrier. Transmigration also seemed to cause "a marked change in the transcriptome of the brain endothelial cells", which basically caused the upregulation of adhesins, integrins and selectins, including ICAM-1, E-selectin, P-selectin and TLR4. Other cytokines whose transcriptions were upregulated include IL6, MCP-1 and the cytokines CCL2, CCL7, CXCL1, CXCL2 and CX3CL1. Since the entry is essentially paracellular, and because the parasite must continue to evade the immune system, they found that certain molecules were downregulated. These included claudin 8, that belongs to the claudin group of proteins that are involved with sealing of the tight junctions. Downregulation of claudin 8 causes loosening of the junctions. Interferon gamma production was downregulated as well, by the downregulation of the transcription factor STAT4. The authors suggest that preliminary evidence exists to support the hypothesis that extracellular tachyzoites are not as capable of crossing the BBB as ones that are infecting leucocytes.
In conclusion, Toxoplasma gondii seems to be a parasite that is more than capable of controlling the host immune response in all the ways necessary to achieve its purposes of entering the brain. Once in the brain, the parasite forms cysts that contain a slowly replicating form (bradyzoites). This causes a latent form of the disease, that in humans occasionally manifests as Toxoplasmic encephalitis or TE. TE patients exhibit neurological symptoms that may include schizophrenia. The exact prognosis of infection depends on various controllable and non-controllable factors that are wonderfully reviewed by Carruthers and Suzuki, 2007. Although it is predominantly an animal parasite, the zoonotic nature of Toxoplasma surely compels our attention.
References in no particular order :
Sanecka A, Frickel EM. Use and abuse of dendritic cells by Toxoplasma gondii. Virulence. 2012 Nov 15;3(7):678-89.Review.
Feustel SM, Meissner M, Liesenfeld O. Toxoplasma gondii and the blood-brain barrier. Virulence. 2012 Mar-Apr;3(2):182-92.
Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O. Intracellular transport of Toxoplasma gondii through the blood-brain barrier. J Neuroimmunol. 2011 Mar;232(1-2):119-30.
Carruthers VB, Suzuki Y. Effects of Toxoplasma gondii infection on the brain. Schizophr Bull. 2007 May;33(3):745-51. Epub 2007 Feb 23. Review.
Galea I, Bechmann I, Perry VH. What is immune privilege (not)? Trends Immunol. 2007 Jan;28(1):12-8.
Denkers EY, Butcher BA, Del Rio L, Bennouna S. Neutrophils, dendritic cells and Toxoplasma. Int J Parasitol. 2004 Mar 9;34(3):411-21. Review.
Barragan A, Brossier F, Sibley LD. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell Microbiol. 2005 Apr;7(4):561-8.
Coisne C, Faveeuw C, Delplace Y, Dehouck L, Miller F, Cecchelli R, Dehouck B. Differential expression of selectins by mouse brain capillary endothelial cells in vitro in response to distinct inflammatory stimuli. Neurosci Lett. 2006 Jan 16;392(3):216-20. Epub 2005 Oct 7.
Lambert H, Vutova PP, Adams WC, Loré K, Barragan A. The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect Immun. 2009 Apr;77(4):1679-88.
"Toxoplasmosis life cycle en" by LadyofHats. Licensed under Public domain via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Toxoplasmosis_life_cycle_en.svg#mediaviewer/File:Toxoplasmosis_life_cycle_en.svg
Just as the space shuttles were designed to carry payloads and people to the low Earth orbit, the zoonotic apicomplexan, Toxoplasma gondii , uses the dendritic cells of the host to move around uninhabitable spaces and cross barriers that it normally cannot by itself cross. Most comparisons call for the dendritic cells to play the role of a Trojan horse, but the space shuttle analogy works just as well.
Here is a brief overview of the life cycle before we scrutinize Toxoplasma's insane abilities to flag down, hitch rides and hijack shuttles. When the infective bradyzoites (in the cyst) are ingested by the definitive host (domestic cat and other Felids), the sexual cycle ensues in the intestines and oocysts are shed in the feces; quite similar to other Coccidians. However, the asexual cycle in the intermediate hosts is what sets Toxoplasma apart. In humans, sheep, mice and every other imaginable host, infective oocysts differentiate into tachyzoites which invade the intestines, multiply and later migrate in the body to form cysts containing bradyzoites. Favorite spots for cyst formation include the brain, the placenta (resulting in abortion(s)) and the eyes; every one of these organs are "immuno-previleged", that is, they are places where innate immuno-surveillance is lower than in the other organs of the body. Before cysts can be formed, Toxoplasma gondii has to get to these places.The migration of a stage called the tachyzoite is the theme of this post.
Getting to know your shuttle
The Dendritic cells are an enigmatic group of leukocytes, first discovered by Steinman R. in 1973, (Steinman was awarded the 2011 Nobel Prize in Physiology/Medicine, only three days after his death. You know of course, that the Prize is usually not awarded posthumously and this was a rare exception made in good faith by the Nobel Prize Committee, whose members were entirely unaware of the death of the laurate) , that have an absolutely incredible range of activities to perform. These bone marrow derived cells, migrate to their temporary homes and keep themselves entertained by sampling their environment, taking up and processing antigens. They are the great controllers of the adaptive immune system, the gatekeepers, if you will, and live throughout the body, at all possible portals of pathogen entry (the skin, the intestines etc), and the secondary lymph organs. They play key roles in immunological tolerance. Once they encounter an antigen that is deemed dangerous, based on its molecular phenotypic profile, or in simple terms the pattern of its dress, they take it up, process it (read "beat it to a pulp") and later migrate with it to lymph nodes where they "present" the processed antigen (on silver platters called MHC molecules) to T cells that are fit to receive such presents (viz., antigen specific). All the while, the DCs secrete chemokines, through which other cells are activated and the immune response spirals into a cell mediated or humoral response as necessary.
DCs exist as distinct subtypes. The jury is out on whether these subtypes are specialized lineages or just functionally plastic cells. The cellular marker of a mature DC in mouse is the CD11c molecule. Interdigitating DCs carry CD205 as well. CD11b is another marker of interest. In mice spleen, CD4-CD8+, CD4+CD8- and CD4-CD8- DC populations exist. Thymic DCs in mice are predominantly CD4-CD8+CD205+CD11b-. Lymph node DCs are predominantly CD4-CD8-CD205-CD11b+ cells. Langerhans DCs dominate the skin draining LNs, and are CD4-CD8-CD205+CD11b+.
Toxoplasma gondii, the perfect parasite, makes use of the migratory activity of this mysterious cell type. In the intermediate host (the animals on the left side on the picture above; that is, mice, humans and other mammals), the parasite enters the body through the mouth when the infective oocyst stage is accidentally consumed. Oocysts that are shed by a household cat is usually the reason that humans come in contact with the parasite. Toxoplasma oocysts unmask themselves to show their virulent side, becoming fast replicating tachyzoites, by invading the intestinal cells and replicating. The DC in the vicinity leap into action. But, even as they lash out, Toxoplasma tachyzoites invade them and causes them to become hypermotile, even in the absence of chemotactic stimuli from other predilection sites. The precise mechanism by which it uses the DCs as its own personal mode of transportation is only slightly known. Within 2 - 3 hrs the parasite rides its shuttle to the local lymph node, each content to bring the other to the LN.
Leaping to action
The DC response at the first hint of the tachyzoites is the production of IL12, triggered through TLR 11 recognition of a profilin-like molecule of the parasite and CCR5 mediated recognition of cyclophilin 18. Other TLRs that are activated include TLR2 and TLR4, both of which are activated by Toxoplasma's glycosylphosphotidylinositol anchors. Once the TLRs are activated, the response is rapid. IL12 activates NK cells, which secrete the first doses of interferon gamma. By this time, the DCs have migrated to the lymph node and present the Profilin-like molecule on MHC Class II molecules. DCs continue to secrete IL12. Antigen specific CD4+ cells are activated by the cytokine mileu and the presented antigen. They begin to pour out IFN gamma in a perfect example of a positive feedback loop, driving their own differentiation down a Th1 road. And you probably know that Th1 responses are primarily cell mediated. Also, DCs are capable of direct antigen presentation on MHC Class I molecules to CD8+ cells, activating them. Actively dividing tachyzoites are now cleared by the CD4+ and CD8+ cells that have migrated to the infection sites in the intestines and the acute phase of the infection ends. The immune system seems to have done its job.
However, the tachyzoites that infected the DCs that have now reached the local lymph node are alive and well. Once a DC reaches the lymph node, it does not ordinarily leave very easily under normal circumstances. Toxoplasma infected DCs though exhibit a bizarrely hypermotile phenotype that allows them to be steered as Toxoplasma pleases. The CD 11c on the surface is down regulated and the CD 11b is upregulated. The infected cells turn from CD11c+CD11b+/- to CD11c-CD11b+.
According to a study by Lambert et al. (2009), hypermotility of the DCs also depends on the genotype of Toxoplasma, with Type II strains showing "stronger intracellular associations with CD11c+ DCs". Types II and III were better at causing DC movement than Type I, although the latter seemed to have better motility on its own, and was able to cross physiological barriers than the former two.
The destination
In an elegant review article, Galea et al. (2006) clarify the 'immune privileged' status experienced by the brain, which is where Toxoplasma controlled DCs are headed. They reiterate that "immune privilege is relative and not absolute", that is, whilst the brain in its normal condition does not host DCs and other antigen presenting cells, an inflammatory response is an altogether different story. One possible reason for this according to other neuroscientists including Coisne et al. 2006 is the "Differential expression of selectins" and other adhesins like ICAM-1 and VCAM-1.
Barragan et al. in 2005 have shown that when Toxoplasma transmigrates, that is, moves across the blood brain barrier without disrupting it, the layers of intervening cells maintain their structure and integrity. And because this was studied in an electrical organ like the brain, they also measured the Transcellular electrical resistance, which they found to be maintained at a steady state during said parasite-DC migration. Using ICAM-1 antibodies, they discovered that ICAM-1 on the host cell surface was involved and that it interacted with a set of micronemal proteins on Toxoplasma called MIC2.
In a more recent study, Lachenmaier et al. (2011) pinned the exact mechanism of neuroinvasion by stating that CD11b+ cells were the main cell population that were used. They further claimed that both type I and II strains were equally capable of infecting CD11b+/CD11c+ cells and migrating across the blood brain barrier. Transmigration also seemed to cause "a marked change in the transcriptome of the brain endothelial cells", which basically caused the upregulation of adhesins, integrins and selectins, including ICAM-1, E-selectin, P-selectin and TLR4. Other cytokines whose transcriptions were upregulated include IL6, MCP-1 and the cytokines CCL2, CCL7, CXCL1, CXCL2 and CX3CL1. Since the entry is essentially paracellular, and because the parasite must continue to evade the immune system, they found that certain molecules were downregulated. These included claudin 8, that belongs to the claudin group of proteins that are involved with sealing of the tight junctions. Downregulation of claudin 8 causes loosening of the junctions. Interferon gamma production was downregulated as well, by the downregulation of the transcription factor STAT4. The authors suggest that preliminary evidence exists to support the hypothesis that extracellular tachyzoites are not as capable of crossing the BBB as ones that are infecting leucocytes.
In conclusion, Toxoplasma gondii seems to be a parasite that is more than capable of controlling the host immune response in all the ways necessary to achieve its purposes of entering the brain. Once in the brain, the parasite forms cysts that contain a slowly replicating form (bradyzoites). This causes a latent form of the disease, that in humans occasionally manifests as Toxoplasmic encephalitis or TE. TE patients exhibit neurological symptoms that may include schizophrenia. The exact prognosis of infection depends on various controllable and non-controllable factors that are wonderfully reviewed by Carruthers and Suzuki, 2007. Although it is predominantly an animal parasite, the zoonotic nature of Toxoplasma surely compels our attention.
References in no particular order :
Sanecka A, Frickel EM. Use and abuse of dendritic cells by Toxoplasma gondii. Virulence. 2012 Nov 15;3(7):678-89.Review.
Feustel SM, Meissner M, Liesenfeld O. Toxoplasma gondii and the blood-brain barrier. Virulence. 2012 Mar-Apr;3(2):182-92.
Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O. Intracellular transport of Toxoplasma gondii through the blood-brain barrier. J Neuroimmunol. 2011 Mar;232(1-2):119-30.
Carruthers VB, Suzuki Y. Effects of Toxoplasma gondii infection on the brain. Schizophr Bull. 2007 May;33(3):745-51. Epub 2007 Feb 23. Review.
Galea I, Bechmann I, Perry VH. What is immune privilege (not)? Trends Immunol. 2007 Jan;28(1):12-8.
Denkers EY, Butcher BA, Del Rio L, Bennouna S. Neutrophils, dendritic cells and Toxoplasma. Int J Parasitol. 2004 Mar 9;34(3):411-21. Review.
Barragan A, Brossier F, Sibley LD. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell Microbiol. 2005 Apr;7(4):561-8.
Coisne C, Faveeuw C, Delplace Y, Dehouck L, Miller F, Cecchelli R, Dehouck B. Differential expression of selectins by mouse brain capillary endothelial cells in vitro in response to distinct inflammatory stimuli. Neurosci Lett. 2006 Jan 16;392(3):216-20. Epub 2005 Oct 7.
Lambert H, Vutova PP, Adams WC, Loré K, Barragan A. The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect Immun. 2009 Apr;77(4):1679-88.
"Toxoplasmosis life cycle en" by LadyofHats. Licensed under Public domain via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Toxoplasmosis_life_cycle_en.svg#mediaviewer/File:Toxoplasmosis_life_cycle_en.svg
Wednesday, June 11, 2014
O live, and make us weep to hear your fate, fair Cryptosporidium, who art smitten by a virus
Act I : Exposition
Imagine a virus that can infect Protists! Although it is common knowledge that viruses are as ubiquitous as other microorganisms, and that each living species probably has a set of viruses that they specifically play host to, protozoan viruses are not exactly virology textbook material. Without exception though, these strange entities can still be classified under the Baltimore system of classification. Trichomonas, Babesia, Giardia, Eimeria, Leishmania and Cryptosporidium, among others, host some of the recently discovered ones. For this blog, I am going to focus on the virus that infects the protozoan Cryptosporidium, for no reason other than that it affects my favorite Apicomplexan.
In 1995, Gallimore and his colleagues, published the first report of a picobirnavirus with an "atypical genome" that they had found in human fecal samples that were positive for Cryptosporidium parvum oocysts. They had detected two fragments of 1.75 and 1.55 Kbp size with Polyacrylamide Gel Electrophoresis. They found the genome "atypical" because it was considered far too small in comparison with previously described "typical" picobirnaviruses.
If you know how naming in virology works, you probably have deduced that the virus under scrutiny is small and has two segments in its RNA genome.
Act II: Rising action
The initial description was followed by others. A publication by Khramtsov et al. in 1997, shows how researchers attempted to characterize the virus. They found that the "novel" extrachromosomal dsRNA that they had isolated from the oocysts of Cryptosporidium parvum was found only in that species (viz. C. parvum), but was absent in other "non-parvum" species that they tested. I found the "non-parvum" species very interesting indeed! C. serpentis of snakes, C. andersoni of cattle, C. baileyi of birds, C. muris of mice and an ostrich isolate were the ones they had tested. Since the ostrich fecal isolate used cannot be identified with any degree of certainty, I offer no comments on it. Other than the odd-ball C. baileyi however, the other three species belong to the gastric group of Cryptosporidium, whereas C.parvum belongs to the intestinal group. So, it would be fairly interesting to see if both groups harbour the virus and if the other intestinal species actually lack it entirely.
Act III: Climax
Khramtsov et al., also found that the larger of the two segments after cloning proved to be a single open reading frame that encoded a protein that was homologous to a virally coded RNA dependent RNA polymerase and that the smaller one was homologous to a MAP kinase. The RNAs were also susceptible to degradation by RNAse A, possibly because they were unencapsidated.
In 2000, Khramtsov et al., isolated the ds RNA from both the calf and human isolates of Cryptosporidium parvum sensu lato, which were then known as C.parvum genotype II and C.parvum genotype I respectively. We know now that the human isolates were in fact C. hominis. Thus, our question must be modified. Can intestinal species other than the ones we now know as C.hominis and C.parvum be infected by the virus?
Subsequent research has found double stranded RNA molecules with 86% similarity in nucleic acid extracts from C.felis and C.meleagridis, both of which most definitely belong to the intestinal group.
Act IV: Falling action
In 2004, Kniel et al., had the absolutely brilliant idea of utilizing the Cryptosporidium parvum virus (CPV; not to be confused with Canine Parvovirus or Cowpox virus) antigen as a target for the detection of C. parvum oocysts. SDS-PAGE analysis of the CPV antigen showed that it was a 40kDa protein. However, for reasons unexplored, the anti- recombinant CPV antibodies raised in rabbits cross-reacted with another 30 kDa protein as well. Immunofluorescence indicated that the antigen localized at the apical end of the parasite and once again no one knows why. The claim of the principle to remarkable sensitivity is well supported by the fact that one single oocyst suspended in water could be identified using an immunoblot assay. However, the long-lasting CPV antigen, which is not degraded even after 3 months, cannot be used to differentiate infectious oocysts from non-infectious ones, which severely dampened the enthusiasm of diagnosticians.
Further research along the same lines was carried out in 2008 by Jenkins et al. The near flawless principle quickly deteriorated because of the sheer complexity of protein folding. The researchers made monoclonal Abs against the 40 kDa protein, because they thought a hybridoma would provide "an unlimited source of detection reagent" and increase assay sensitivity. But, despite using an indirect IFA, the sensitivity was not much higher than the conventional IFA that targets the outer wall protein. Nevertheless, on a dot blot assay, the sensitivity was seemingly incredible. As low as 100 non-bleach treated oocysts could be detected. The authors however agree that this jaw dropping oddity was probably due to contamination and the binding of MAbs by Fc receptors on contaminant bacteria, because bleach treated oocysts (which are standards for research), could not be detected well. Another wall that the researchers ran up against was the native confirmation of CPV40, which did not allow the binding of the antibody made against the recombinant antigen. It is possible that the recombinant protein made in E.coli did not fold the same way due to lack of eukaryotic chaperone proteins. To circumvent the binding issue, the proteins had to be extracted under denaturing conditions. Under non-denaturing conditions, yet another eccentricity was observed. The MAb could bind to proteins of size 77 and 125 kDa respectively, which were presumed to be viral capsid dimers and tetramers. This presumption begs to be challenged. If the epitope is not exposed in the monomeric capsid, then it surely must not be exposed in the dimeric or higher forms given the regularity and symmetry of the virus. In the end, the detection system is still not fully ready for use.
Act V: Denoument
That same year , the executive committee of International Committee on the Taxonomy of Viruses agreed to formally recognize the virus, which became known as 'Cryptosporidium parvum virus 1' (CSpV1), and establish a new genus called Cryspovirus in the family Partitiviridae, in which it is currently ensconced.
A short paper by Jenkins et al., published in 2008 (Was 2008 the year of the Cryspovirus?) sought to give the virus a role. "Fecundity" , that is , oocyst producing ability of two strains of C. parvum were compared, one of which had the virus and one of which did not. Calves infected with C. parvum with virus had a 5 fold higher oocyst output in their feces compared to the calves infected with C.parvum without the virus. Calf immunity and other variables cast doubts on the report. Does the virus affect the replication of the parasite (given that it is only cytoplasmic) or is the proposal just a fanciful conjecture?
A description paper was later published, cementing the name and the taxonomy. CSpV1 shows vertical transmission only. The virions are isometric, have a diameter of 31nm with short protrusions on their surface.
The story of the Cryspovirus continues to unfold. We are yet to come to a full understanding of its significance in the life history of Cryptosproridium and hence its importance to us. I'm looking forward to future publications!
References :
Gallimore CI, Green J, Casemore DP, Brown DW. Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans. Arch Virol. 1995;140(7):1275-8.
Khramtsov NV, Woods KM, Nesterenko MV, Dykstra CC, Upton SJ. Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum. Mol Microbiol. 1997 Oct;26(2):289-300.
Khramtsov NV, Chung PA, Dykstra CC, Griffiths JK, Morgan UM, Arrowood MJ, Upton SJ. Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum. J Parasitol. 2000 Apr;86(2):275-82.
Leoni F, Gallimore CI, Green J, McLauchlin J. Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis. Parasitol Int. 2006 Dec;55(4):299-306.
Kniel KE, Higgins JA, Trout JM, Fayer R, Jenkins MC. Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts. J Microbiol Methods. 2004 Aug;58(2):189-95.
Jenkins MC, O'Brien CN, Trout JM. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein. J Parasitol. 2008 Feb;94(1):94-8.
Nibert ML, Woods KM, Upton SJ, Ghabrial SA. Cryspovirus: a new genus of protozoan viruses in the family Partitiviridae. Arch Virol. 2009;154(12):1959-65.
Jenkins MC, Higgins J, Abrahante JE, Kniel KE, O'Brien C, Trout J, Lancto CA, Abrahamsen MS, Fayer R. Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV. Int J Parasitol. 2008 Jul;38(8-9):1051-5.
Imagine a virus that can infect Protists! Although it is common knowledge that viruses are as ubiquitous as other microorganisms, and that each living species probably has a set of viruses that they specifically play host to, protozoan viruses are not exactly virology textbook material. Without exception though, these strange entities can still be classified under the Baltimore system of classification. Trichomonas, Babesia, Giardia, Eimeria, Leishmania and Cryptosporidium, among others, host some of the recently discovered ones. For this blog, I am going to focus on the virus that infects the protozoan Cryptosporidium, for no reason other than that it affects my favorite Apicomplexan.
In 1995, Gallimore and his colleagues, published the first report of a picobirnavirus with an "atypical genome" that they had found in human fecal samples that were positive for Cryptosporidium parvum oocysts. They had detected two fragments of 1.75 and 1.55 Kbp size with Polyacrylamide Gel Electrophoresis. They found the genome "atypical" because it was considered far too small in comparison with previously described "typical" picobirnaviruses.
If you know how naming in virology works, you probably have deduced that the virus under scrutiny is small and has two segments in its RNA genome.
(Image from EMDB - 1459)
The initial description was followed by others. A publication by Khramtsov et al. in 1997, shows how researchers attempted to characterize the virus. They found that the "novel" extrachromosomal dsRNA that they had isolated from the oocysts of Cryptosporidium parvum was found only in that species (viz. C. parvum), but was absent in other "non-parvum" species that they tested. I found the "non-parvum" species very interesting indeed! C. serpentis of snakes, C. andersoni of cattle, C. baileyi of birds, C. muris of mice and an ostrich isolate were the ones they had tested. Since the ostrich fecal isolate used cannot be identified with any degree of certainty, I offer no comments on it. Other than the odd-ball C. baileyi however, the other three species belong to the gastric group of Cryptosporidium, whereas C.parvum belongs to the intestinal group. So, it would be fairly interesting to see if both groups harbour the virus and if the other intestinal species actually lack it entirely.
Act III: Climax
Khramtsov et al., also found that the larger of the two segments after cloning proved to be a single open reading frame that encoded a protein that was homologous to a virally coded RNA dependent RNA polymerase and that the smaller one was homologous to a MAP kinase. The RNAs were also susceptible to degradation by RNAse A, possibly because they were unencapsidated.
In 2000, Khramtsov et al., isolated the ds RNA from both the calf and human isolates of Cryptosporidium parvum sensu lato, which were then known as C.parvum genotype II and C.parvum genotype I respectively. We know now that the human isolates were in fact C. hominis. Thus, our question must be modified. Can intestinal species other than the ones we now know as C.hominis and C.parvum be infected by the virus?
Subsequent research has found double stranded RNA molecules with 86% similarity in nucleic acid extracts from C.felis and C.meleagridis, both of which most definitely belong to the intestinal group.
Act IV: Falling action
In 2004, Kniel et al., had the absolutely brilliant idea of utilizing the Cryptosporidium parvum virus (CPV; not to be confused with Canine Parvovirus or Cowpox virus) antigen as a target for the detection of C. parvum oocysts. SDS-PAGE analysis of the CPV antigen showed that it was a 40kDa protein. However, for reasons unexplored, the anti- recombinant CPV antibodies raised in rabbits cross-reacted with another 30 kDa protein as well. Immunofluorescence indicated that the antigen localized at the apical end of the parasite and once again no one knows why. The claim of the principle to remarkable sensitivity is well supported by the fact that one single oocyst suspended in water could be identified using an immunoblot assay. However, the long-lasting CPV antigen, which is not degraded even after 3 months, cannot be used to differentiate infectious oocysts from non-infectious ones, which severely dampened the enthusiasm of diagnosticians.
Further research along the same lines was carried out in 2008 by Jenkins et al. The near flawless principle quickly deteriorated because of the sheer complexity of protein folding. The researchers made monoclonal Abs against the 40 kDa protein, because they thought a hybridoma would provide "an unlimited source of detection reagent" and increase assay sensitivity. But, despite using an indirect IFA, the sensitivity was not much higher than the conventional IFA that targets the outer wall protein. Nevertheless, on a dot blot assay, the sensitivity was seemingly incredible. As low as 100 non-bleach treated oocysts could be detected. The authors however agree that this jaw dropping oddity was probably due to contamination and the binding of MAbs by Fc receptors on contaminant bacteria, because bleach treated oocysts (which are standards for research), could not be detected well. Another wall that the researchers ran up against was the native confirmation of CPV40, which did not allow the binding of the antibody made against the recombinant antigen. It is possible that the recombinant protein made in E.coli did not fold the same way due to lack of eukaryotic chaperone proteins. To circumvent the binding issue, the proteins had to be extracted under denaturing conditions. Under non-denaturing conditions, yet another eccentricity was observed. The MAb could bind to proteins of size 77 and 125 kDa respectively, which were presumed to be viral capsid dimers and tetramers. This presumption begs to be challenged. If the epitope is not exposed in the monomeric capsid, then it surely must not be exposed in the dimeric or higher forms given the regularity and symmetry of the virus. In the end, the detection system is still not fully ready for use.
Act V: Denoument
That same year , the executive committee of International Committee on the Taxonomy of Viruses agreed to formally recognize the virus, which became known as 'Cryptosporidium parvum virus 1' (CSpV1), and establish a new genus called Cryspovirus in the family Partitiviridae, in which it is currently ensconced.
A short paper by Jenkins et al., published in 2008 (Was 2008 the year of the Cryspovirus?) sought to give the virus a role. "Fecundity" , that is , oocyst producing ability of two strains of C. parvum were compared, one of which had the virus and one of which did not. Calves infected with C. parvum with virus had a 5 fold higher oocyst output in their feces compared to the calves infected with C.parvum without the virus. Calf immunity and other variables cast doubts on the report. Does the virus affect the replication of the parasite (given that it is only cytoplasmic) or is the proposal just a fanciful conjecture?
A description paper was later published, cementing the name and the taxonomy. CSpV1 shows vertical transmission only. The virions are isometric, have a diameter of 31nm with short protrusions on their surface.
The story of the Cryspovirus continues to unfold. We are yet to come to a full understanding of its significance in the life history of Cryptosproridium and hence its importance to us. I'm looking forward to future publications!
References :
Gallimore CI, Green J, Casemore DP, Brown DW. Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans. Arch Virol. 1995;140(7):1275-8.
Khramtsov NV, Woods KM, Nesterenko MV, Dykstra CC, Upton SJ. Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum. Mol Microbiol. 1997 Oct;26(2):289-300.
Khramtsov NV, Chung PA, Dykstra CC, Griffiths JK, Morgan UM, Arrowood MJ, Upton SJ. Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum. J Parasitol. 2000 Apr;86(2):275-82.
Leoni F, Gallimore CI, Green J, McLauchlin J. Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis. Parasitol Int. 2006 Dec;55(4):299-306.
Kniel KE, Higgins JA, Trout JM, Fayer R, Jenkins MC. Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts. J Microbiol Methods. 2004 Aug;58(2):189-95.
Jenkins MC, O'Brien CN, Trout JM. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein. J Parasitol. 2008 Feb;94(1):94-8.
Nibert ML, Woods KM, Upton SJ, Ghabrial SA. Cryspovirus: a new genus of protozoan viruses in the family Partitiviridae. Arch Virol. 2009;154(12):1959-65.
Jenkins MC, Higgins J, Abrahante JE, Kniel KE, O'Brien C, Trout J, Lancto CA, Abrahamsen MS, Fayer R. Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV. Int J Parasitol. 2008 Jul;38(8-9):1051-5.
Friday, May 2, 2014
If you gotta start somewhere, why not here - A vaccine for Cytauxzoon felis
One major group that has parasites of medical and veterinary importance is the Subclass Hematozoa of the phylum Apicomplexa. This subclass has 2 orders, one of which has the infamous intraerythrocytic genera - Plasmodium, Leucocytozoon and Hemoproteus, and the other has the Piroplasm genera Theileria and Babesia. (see my previous post to see the relationship between these)
Parasites and vectors - Two worlds collide
While Theileria is the more famous of the parasites in the Family Theileriidae, a new cousin parasite was identified in 1976 in domestic cats in the southern US. Cytauxzoon felis, whose definitive host is the Lynx rufus (Bobcat), causes an acute, lethal disease in Felis domesticus . Few survive. Fantastic reviews on the clinical manifestation are found here and here. Cytauxzoon is vector borne and carried by the lone star tick Amblyomma americanum. Transmission is indirect and prevention has relied on disrupting the life cycle at the tick stage. There have been no traditional vaccines so far, because the piroplasms cannot be grown in culture. It has been found previously that of the three life cycle stages of the parasite, viz., sporozoites, schizonts and merozoites, protective immunity is seen with the schizont stage. Cats that had survived the schizogenous phase of the disease were the rare ones that survived a challenge with the organism again. Merozoites do not protect against infection.
(Source : CDC ; http://www.cdc.gov/stari/disease/)
If we gotta start somewhere, I say here
In a recent article published in PLoS One in Oct 2013, Tarigo J. et al. have used the molecular genomics approach to expedite the identification of a new protein antigen to serve as a vaccine candidate. They started with blood from a cat that had died of C.felis. They isolated the parasitic merozoites from the erythrocytes (RBCs) and sequenced the 9.1 megabase genome using Roche's 454 pyrosequencing.
Next Generation Sequencing is all the rage these days. Sanger's first generation method is near-obsolete.But, you can still get a short product sequenced (using the method) for as low as $5. Roche's 454 pyrosequencing, Illumina's reversible terminator sequencing, ABI's SOLiD sequencing by ligation and Helicos' single molecule sequencing , which are/were the second gen, are being supplanted slowly even as I write by third generation technologies, viz., PacBio Real Time single molecule sequencing, Complete Genomics' combined proanchor hybridization and ligation, and Life Tech's Ion Torrent sequencing. (Hui, 2014).
Now, if you have never been a part of a genome sequencing project, you could be under the incorrect impression that the 454 sequencer spits out perfectly ordered nucleotide data that can be uploaded to GenBank and analysed at leisure. The reality is, however, disturbingly far from such an assumption. You typically get about a hundred thousand (or more) short 'reads' as output. Imagine someone cutting up the front page of a newspaper into individual alphabets and then asking you to reassemble the little pieces into coherent words, sentences, paragraphs and sections. You would think that the person was crazy because the task is near impossible manually. What if you were given a software that could do it for you? With reasonable error, the software could probably do it. Such software that handle genomic data are called assemblers. They put nucleotide data together into cogent contigs de novo, i.e. they form sentences in a new language that they do not know. Some of the most popular of these (for 454 and other platforms) are Newbler, Edena, EULER- SR, Gapcloser, Oases, Ray, SOAPdenovo, Vcake, velvet and wgs. For C.felis genome assembly, the authors used Newbler (Tarigo, 2013; Kumar, 2010)
To study genes that code proteins, transcribed messenger RNA is usually utilized to construct a cDNA library. Traditional cDNA library construction involves a polyT primer sequence and two PCR runs to make double stranded cDNA, followed by cloning into vectors. Improved methods have now appeared on the scene including Clontech's SMARTer PCR cDNA synthesis kit, which is a single step reverse transcription reaction using a 'Switching Mechanism At the 5' end of the RNA Template'. SMART, indeed! The cDNA library was then sequenced to generate ESTs or Expressed Sequence Tags using the 454 platform and Newbler. ESTs, in the newspaper analogy, would be the equivalent of the first and the last few words of a sentence. Since the complete genome was available, ESTs were used to map protein-coding regions back onto the full length genome using other software. In this paper, GeneMark-ES 2.5 was used. Other software that can do the same are Apollo, Augustus, CEGMA, ESTScan, Eval, FrameDP, Genethreader and PASA. These are basic annotation software that model gene structures onto genome data. After the first proteome was computed, a second predicted proteome was created using GlimmerHMM. The data was then used to run BLAST searches on NCBI, focusing specifically on the already deposited transcriptomes of Theileria parva, Babesia bovis and Plasmodium falciparum.
Basic Local Alignment Search Tool for proteins (BLASTp) was then used to predict shared genes. The authors accepted 60% similarities across at least 30% of the query sequence as matches. They identified the cf67 gene on the genome of C.felis, which is syntenic to p67/SPAG-1/BOV57 on Theileria parva. Going back to the newspaper analogy, syntenous genes are equivalent to words that occur at the same place in a sentence. For example, cf67 and p67 are like the fourth word, on a headline spanning ten words, from two different newspapers - they are located at the same spot but are not the same. In the case of cf67 and p67, the three genes/ words preceding them were conserved/the same.The gene was then directionally cloned into a plasmid vector, and the product was used to perform in vitro transcription and translation reactions. The His tagged translation products were purified using Ni-NTA magnetic agarose beads and their quality assessed using western blot analysis with anti-His antibodies. Another western blot was carried out with serum from cats that had been infected but had survived the infection. There was a high degree of immunoreactivity observed with the sera against the C terminal and the whole ORF clones of cf67. This makes cf67 a great candidate antigen for immunological studies.
The authors also found that cf67 is conserved in samples of C.felis from eight states in the southern US, with very little variation at the C terminal region. The cf67 was expressed in the schizogony stage. This was determined with insitu hybridization using a negative sense riboprobe directed against cytoplasmic mRNA in the schizont stage.Thus, the authors were able to identify a highly conserved sequence that was transcribed into an antigenic protein at the lifestage that could be targeted by the immune system to provide protective immunity. The heavy reliance on bioinformatics and in silico genomics is remarkable.
In a related review, Sirskyj et al., labelled such new innovative uses of bioinformatics for vaccine design as 'vaccinomics'. Conventional methods often miss proteins that are expressed at certain life cycle stages, but not at others, and so are potentially missed and overlooked as vaccine candidates. Software are available to predict immunodominant T and B cell epitopes. But despite the best predictions, field and clinical trials have not always been successful (Sirskyj,2011).
There is hope in the distance
Translational medicine techniques, to make the jump form epitope prediction to commercially viable vaccine, are still lacking across the veterinary and human medical and research fields. Although safety and efficacy are big concerns with conventional cell derived vaccines, they are not so with recombinant protein vaccines. Immune responses are slightly more controlled with the latter. The biggest concerns however arise from the degradation of the vaccine (either the insert or the vector) inside the body, lack of an effective strong immunity and delivery routes. These issues are more easily overcome in the veterinary field than in the human field because of sanctions on testing of vaccine candidates on animals. So far, the licensed recombinant vaccines available in the US for animals include the gI and gX deleted DIVA vaccine for pseudorabies in pigs and 11 poultry vaccines including fowlpox vectored vaccines for avian influenza, ND, Avian encephalomyelitis, ILT and Mycoplasma gallisepticum; Marek's disease virus vectored vaccine for ND, ILT deletion mutants of Salmonella typhimurium and deletion mutant of ILTV expressing HA from avian influenza.Vaccines for companion animals include canarypox vectored canines vaccines against Lyme disease, CDV and rabies; canary pox vectored feline vaccines against FeLV and rabies. Vaccines for horses include canary pox vectored vaccines against west nile virus and equine influenza. The newly identified vaccine candidate antigen for Cytauxzoon felis needs a lot of dedicated labwork ex silico before it can be made into a commercial vaccine for your pet. But in silico predictions are a great way to start.
References :
Tarigo JL, Scholl EH, McK Bird D, Brown CC, Cohn LA, Dean GA, Levy MG, Doolan DL, Trieu A, Nordone SK, Felgner PL, Vigil A, Birkenheuer AJ. A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics. PLoS One. 2013 Aug 20;8(8):e71233
Kumar S, Blaxter ML. Comparing de novo assemblers for 454 transcriptome data. BMC Genomics. 2010 Oct 16;11:571.
Hui P. Next generation sequencing: chemistry, technology and applications. Top Curr Chem. 2014;336:1-18.
Sirskyj D, Diaz-Mitoma F, Golshani A, Kumar A, Azizi A. Innovative bioinformatic approaches for developing peptide-based vaccines against hypervariable viruses. Immunol Cell Biol. 2011 Jan;89(1):81-9.
Tuesday, April 8, 2014
A midsummer nightmare - Cyclospora
In the mid summer of 2013, 631 people across 25 states in the US became ill with diarrhea, anorexia, low grade fever, nausea, and other non specific gastrointestinal symptoms. About 49 of these patients had symptoms severe enough to warrant hospitalization. A mad scramble of trace-back investigations in Iowa and Nebraska traced the outbreak to two famous restaurant chains and the consumption of salads that originated outside the country. A curious coccidian parasite called Cyclospora cayetanensis was identified as the etiological agent.
Before we analyse the associated investigation, let us look at the parasite itself :
Before we analyse the associated investigation, let us look at the parasite itself :
Cyclospora was first recognized in the early 1980s in patients with enteric disease. 'Enteric disease' is an elegant medical term that encompasses the whole spectrum of clinical signs (nausea, diarrhea, abdominal pain, cramps etc) that are manifested by pathological changes in the small and large intestines. Cyclospora was initially misdiagnosed as everything ranging from artifacts to blue green alga, with the range in between including Cryptosporidium, Isospora, Coccidian-like body, cyanobacterium-like body, and unidentified flagellate. Reports in 1995 put an end to speculation by characterizing the SSU rRNA gene and concluding that the parasite was related to Eimeria , which as you all know is a Coccidian apicomplexan.
Cyclospora sps. are parasites of a wide range of snakes, rodents and mammals, not necessarily in that order, and they appear to be monoxenic (that is to say, they complete all parts of their lifecycle in one host). As is tradition, the oocysts are of diagnostic importance and are fairly typical. They have two sporocysts with two sporozoites each. Each of the sporozoites is folded in two. Diagnostic tests that detect the parasite are in fact designed to recognize this stage. These include the classic ones including the acid fast staining (picture below) with bright field microscopy, modified safranin staining, trichrome staining and the unconventional UV epifluorescence microscopy. Jejunal biopsies and PCR amplification of target genes can detect other stages besides the oocyst stage, which is shown below.
(Source : http://phil.cdc.gov/Phil/details.asp )
A typical Coccidian life cycle
Oocysts are excreted unsporulated in the feces of the infected host. They sporulate in 7 -15 days and become infectious. The sporulated oocysts ultimately come to be found on the surfaces of green leafy vegetables and fruits. Upon ingestion by a new host, the oocysts excyst, infect the columnar epithelial cells of the proximal intestines (viz, duodenum and jejunum) and undergo merogony to produce two types of meronts that differentiate and undergo gametogony. The macro and microgametocytes fuse to form oocysts that are excreted.
Public health concerns
Evaluating the parasite from the perspective of the epidemiological triad brings out the following:
Parasite factors : Long sporulation time (7-15 days), which precludes direct person-to-person transmission; resistance to common disinfectants including chlorine; surface adhesins confer strong attachment to fresh produce.
Host factors : So far, Cyclospora cayetanensis seems to infect only humans, although oocysts have been isolated in fowl and dogs in endemic countries. But, the exact role of intermediate and transport hosts is yet to be elucidated, as is also the role of Cyclospora as an opportunistic pathogen. Undoubtedly, infections in immunocompromised patients are symptomatic, especially when the immunocompromise is due to infection with that devious Lentivirus called the Human Immunodeficiency Virus. A 2003 study in Venezuela reported a high percentage of asymptomatic carriers among the other major target population viz. infants.
Animal models have been unsuccessful so far.
Environmental factors : Microenvironmental factors that dictate sporulation are not entirely unknown. Temperature between 22C and 32C is needed for sporulation, which confines the circum-annual distribution to the tropics. This also means that the hemisphere experiencing summer at any given time is more prone to outbreaks than its antipodes. Exact seasonality and geographical distribution are under study. This predominantly tropical disease, has led to outbreaks in temperate regions as well due to the importation of fresh produce. Travelers to endemic areas tend to pick up the infection due to sheer lack of knowledge of the existence of a parasite of this sort. A broad knowledge base and smart travel skills have become more important than ever. Cases in the United States are sporadic, of which some are acquired internationally and others domestically.
The map below shows cases that were acquired domestically between 1997 and 2008 :
(Source : cdc.gov/mmwr/preview/mmwrhtml/ss6002a1.htm)
The 2013 outbreak
The summer 2013 outbreak peaked between the 16th and 22th of June, and the second spike occurred between the 30th of June and the 6th of July, with 174 and 111 cases respectively.The five-year incidence mean for the same two weeks are 6.8 and 5.6 cases respectively.
The FDA conducted an Environmental Assessment as part of the response to the outbreak. Investigations were conducted between the 12th and the 19th Aug, 2013, more than five weeks after the last date that patients had eaten at one of the restaurants affected ( i.e. July 2, 2013). (Read the FDA report to obtain restaurant names). The salad mix supplied to the two restaurants was produced by T Farms, in a neighboring country. The salad had contained iceberg lettuce, romaine lettuce, green leaf, red cabbage and carrots, of which only the latter was processed at a different facility.
The processing facility was first investigated in true Sherlock style. Construction and design of the plant,
water processing system, sanitary water system, receipt of raw ingredients, employee health and distribution systems were assessed. These were considered potential sources of the parasite. Infrastructural items and washwater obtained after vegetable washing were systematically cleared and proven negative by standard lab techniques (microscopy and PCR) as the source of the infection.
The source of raw ingredients were assessed in the "ranches" on which they were grown. A thorough examination of the employees working on these ranches and their medical records proved that they were not the source of infection. An assessment of the wells and farms in the vicinity of the ranches proved that they were not sources of infection either. Topography of the land and the rainfall patterns indicated that those geological/meteorological entities did not contribute to the outbreak. The EA team deemed other things on the ranches to be fairly in order and suggested a few minor modifications to the irrigation systems, such as the installation of back flow pumps to wells and the changing of valves on sinks that could potentially lead to cross-contamination.
The final conclusion offered is, " the FDA has not been able to definitively determine how or at what point in the supply chain Cyclospora cayetanensis contaminated the salad mix associated with the outbreak."
Before you criticize or condemn the lack of an expected conclusion, remember that epidemiological investigations are not easy. They do not always conclude successfully. That is, what you want to find is not what you will find. This is true of the biological sciences in general. Hypotheses have to be assessed and changed as one proceeds with data collection. To steadfastly hold onto a pretentious hypothesis and to try and fit the data to your hypothesis is obviously the hallmark of one working with a closed mind. As for Cyclospora, the matter rests for now, atleast until the next food associated outbreak.
References :
Environmental Assessment: 2013 Cyclosporiasis outbreak in Iowa and Nebraska – Findings and Recommendations
http://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ucm375732.htm
Cyclospora sps. are parasites of a wide range of snakes, rodents and mammals, not necessarily in that order, and they appear to be monoxenic (that is to say, they complete all parts of their lifecycle in one host). As is tradition, the oocysts are of diagnostic importance and are fairly typical. They have two sporocysts with two sporozoites each. Each of the sporozoites is folded in two. Diagnostic tests that detect the parasite are in fact designed to recognize this stage. These include the classic ones including the acid fast staining (picture below) with bright field microscopy, modified safranin staining, trichrome staining and the unconventional UV epifluorescence microscopy. Jejunal biopsies and PCR amplification of target genes can detect other stages besides the oocyst stage, which is shown below.
(Source : http://phil.cdc.gov/Phil/details.asp )
A typical Coccidian life cycle
Oocysts are excreted unsporulated in the feces of the infected host. They sporulate in 7 -15 days and become infectious. The sporulated oocysts ultimately come to be found on the surfaces of green leafy vegetables and fruits. Upon ingestion by a new host, the oocysts excyst, infect the columnar epithelial cells of the proximal intestines (viz, duodenum and jejunum) and undergo merogony to produce two types of meronts that differentiate and undergo gametogony. The macro and microgametocytes fuse to form oocysts that are excreted.
(Source : http://www.cdc.gov/parasites/cyclosporiasis/biology.html)
Public health concerns
Evaluating the parasite from the perspective of the epidemiological triad brings out the following:
Parasite factors : Long sporulation time (7-15 days), which precludes direct person-to-person transmission; resistance to common disinfectants including chlorine; surface adhesins confer strong attachment to fresh produce.
Host factors : So far, Cyclospora cayetanensis seems to infect only humans, although oocysts have been isolated in fowl and dogs in endemic countries. But, the exact role of intermediate and transport hosts is yet to be elucidated, as is also the role of Cyclospora as an opportunistic pathogen. Undoubtedly, infections in immunocompromised patients are symptomatic, especially when the immunocompromise is due to infection with that devious Lentivirus called the Human Immunodeficiency Virus. A 2003 study in Venezuela reported a high percentage of asymptomatic carriers among the other major target population viz. infants.
Animal models have been unsuccessful so far.
Environmental factors : Microenvironmental factors that dictate sporulation are not entirely unknown. Temperature between 22C and 32C is needed for sporulation, which confines the circum-annual distribution to the tropics. This also means that the hemisphere experiencing summer at any given time is more prone to outbreaks than its antipodes. Exact seasonality and geographical distribution are under study. This predominantly tropical disease, has led to outbreaks in temperate regions as well due to the importation of fresh produce. Travelers to endemic areas tend to pick up the infection due to sheer lack of knowledge of the existence of a parasite of this sort. A broad knowledge base and smart travel skills have become more important than ever. Cases in the United States are sporadic, of which some are acquired internationally and others domestically.
The map below shows cases that were acquired domestically between 1997 and 2008 :
(Source : cdc.gov/mmwr/preview/mmwrhtml/ss6002a1.htm)
The 2013 outbreak
The summer 2013 outbreak peaked between the 16th and 22th of June, and the second spike occurred between the 30th of June and the 6th of July, with 174 and 111 cases respectively.The five-year incidence mean for the same two weeks are 6.8 and 5.6 cases respectively.
The FDA conducted an Environmental Assessment as part of the response to the outbreak. Investigations were conducted between the 12th and the 19th Aug, 2013, more than five weeks after the last date that patients had eaten at one of the restaurants affected ( i.e. July 2, 2013). (Read the FDA report to obtain restaurant names). The salad mix supplied to the two restaurants was produced by T Farms, in a neighboring country. The salad had contained iceberg lettuce, romaine lettuce, green leaf, red cabbage and carrots, of which only the latter was processed at a different facility.
The processing facility was first investigated in true Sherlock style. Construction and design of the plant,
water processing system, sanitary water system, receipt of raw ingredients, employee health and distribution systems were assessed. These were considered potential sources of the parasite. Infrastructural items and washwater obtained after vegetable washing were systematically cleared and proven negative by standard lab techniques (microscopy and PCR) as the source of the infection.
The source of raw ingredients were assessed in the "ranches" on which they were grown. A thorough examination of the employees working on these ranches and their medical records proved that they were not the source of infection. An assessment of the wells and farms in the vicinity of the ranches proved that they were not sources of infection either. Topography of the land and the rainfall patterns indicated that those geological/meteorological entities did not contribute to the outbreak. The EA team deemed other things on the ranches to be fairly in order and suggested a few minor modifications to the irrigation systems, such as the installation of back flow pumps to wells and the changing of valves on sinks that could potentially lead to cross-contamination.
The final conclusion offered is, " the FDA has not been able to definitively determine how or at what point in the supply chain Cyclospora cayetanensis contaminated the salad mix associated with the outbreak."
Before you criticize or condemn the lack of an expected conclusion, remember that epidemiological investigations are not easy. They do not always conclude successfully. That is, what you want to find is not what you will find. This is true of the biological sciences in general. Hypotheses have to be assessed and changed as one proceeds with data collection. To steadfastly hold onto a pretentious hypothesis and to try and fit the data to your hypothesis is obviously the hallmark of one working with a closed mind. As for Cyclospora, the matter rests for now, atleast until the next food associated outbreak.
References :
Environmental Assessment: 2013 Cyclosporiasis outbreak in Iowa and Nebraska – Findings and Recommendations
http://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ucm375732.htm
Monday, March 3, 2014
Phylum defining feature : The apical complex
The Phylum Apicomplexa is a large and uniquely diverse collection of unicellular, eukaryotic organisms that share amongst other common features, one that is capable of conferring the honor of being placed in this phylum, and one from which this blog derives its name . Ladies and gentlemen, let me introduce (cue fanfare ♪♫♪), the Apical Complex.
Ontologically, the apical complex is a constituent cellular component, found at the anterior end of the mature parasite. It is made up of various cytoskeletal components and membrane bound organelles, and among the latter , we find the micronemes, rhoptries, polar rings and conoids. (All that is standard textbook fare).
.
Zooming in closer on Toxoplasma gondii, an archetypical apicomplexan, as Hu et al. have done in their paper , we find that the conoid is spirally arranged, which naturally allows for its role in invasion. The conoid is associated with the preconoidal rings, the polar rings and intraconoid microtubules. The polar rings serve as an anchor for microtubules that run from the anterior end of the organism to the posterior end, right under the pellicle. These, because of their location, are called subpellicular microtubules. Microtubule numbers vary across the phylum.When viewed under a phase contrast microscope, the entire apical complex appears as a single dark spot on the anterior end of the parasite.
The conoid is quite remarkable in that although it is made up of alpha-beta tublulins that are similar to the ones found in mammals, the tubulins form an unique ribbon-like polymer. When calcium levels in the cell rise, the conoid along with the associated upper polar ring protrudes beyond the base of the lower polar ring. This is shown in these figures on PubMed, in which alpha-tublin was modified with yellow fluorescent protein labelling.
The micronemes are involved in attachment and penetration. The proteins, named MICs, in these organelles are secreted, before the exocytosis of the rhoptry proteins. Before they get to their compartment, these proteins which have a classic hydrophobic amino terminal signal peptide move through the endoplasmic reticulum and exit the network at the golgi complex. They additionally have two conserved motifs at the C terminus - the first being a SYHYY and the second, an acidic series, EIEYE. These are important in folding, sorting and the spanning of the membrane. There are also distinct adhesive domains in the middle.
As soon as the tachyzoite attaches to the host cell, there is a marked increase in calcium levels in the parasitic cell, as calcium from the acidocalcisomes is released. This triggers the activation of at least two calcium dependant protein kinases that cause further signal cascades, and ultimately the release of micronemal proteins. The adhesive domains are involved in the adhesion and further invasion may take place.
Rhoptry proteins, reviewed in an article in Nature Reviews Microbiology,2008 , by Boothroyd, is conserved in its presence across the phylum and yet is varied in its architecture and numbers. There are 12 in the tachyzoite stages of Toxoplasma, each 2 to 3 micrometers in length. They appear to have compartments when studied with IFA. Protein compartmentalization post-transcriptionally occurs by understudied mechanisms. Rhoptries are even visible under a phase contrast microscope and appear to be related to exosomes. Of the 29 proteins identified in the rhoptries, 24 ROP proteins localize in the bulb region and 5 RON proteins localize in the neck region, with 28 newly recognized proteins yet to be allotted designations/names. Some of the ROP proteins are enzymes like kinases, proteases and phosphatases, that do not have orthologs in related genera. The, one gene- one protein, RON proteins however, have orthologs in related extant sps, although their exact functions are yet to be fully elucidated. The rhoptries also seem to possess lipids rich in cholesterol. These are suspected to play a role during invasion, by forming vesicles around secreted proteins and ultimately fusing with the parasitophorous vacuole (PV).
During cellular invasion, the rhoptry contents are released. Some of the RON proteins are involved in the formation of the moving junction, which forms the physical interface, that is , the point of contact during invasion. The ROP proteins, after their release, localize in the PV or the PV membrane (PVM) or inside the host cell (including the nucleus), with many other targets being yet unknown. ROP1 is released in the PV as a vesicle and later moves to the membrane. ROP2 and its associates are putative integral PVM proteins that recruit the host mitochondria to the cytoplasmic face of the PVM. PP2C-hn and ROP16 contain typical NLS (Nuclear Localization Signal) sequences that cause them to move to the nucleus. All these molecules seem to play a key role in the biology of Toxoplasma sps.
The exact mechanism of conservation of the apical complex across the phylum seems to be a curious thing, as the phylum seems to be inhabited by protozoans that are as different, from each other in their pathogenesis and habitat, as the night is from the day. It certainly is true that the use of the apical complex is not always the same in all species, and certainly not all have the above described mechanism of Toxoplasma gondii. Thus, the Toxoplasma model, despite being the best that we have got right now, gives us only a glimpse of the preeminence of the great phylum-defining feature. It surely is our starting point. Where we will yet reach from here is unknown . Will we ravel the marvels of the pathogenesis of, say Cyclospora, by studying this feature? Will we bring to light the reason for the bizzare behaviour of the other Apicomplexans ? We can only hope so, for now, because, we have miles to sail in our research before we reach the shores of understanding .
References :
Ontologically, the apical complex is a constituent cellular component, found at the anterior end of the mature parasite. It is made up of various cytoskeletal components and membrane bound organelles, and among the latter , we find the micronemes, rhoptries, polar rings and conoids. (All that is standard textbook fare).
.
(Source: http://tolweb.org/tree/ToLimages/zoite-tolv1.png; under the Creative Commons License)
The conoid is quite remarkable in that although it is made up of alpha-beta tublulins that are similar to the ones found in mammals, the tubulins form an unique ribbon-like polymer. When calcium levels in the cell rise, the conoid along with the associated upper polar ring protrudes beyond the base of the lower polar ring. This is shown in these figures on PubMed, in which alpha-tublin was modified with yellow fluorescent protein labelling.
The micronemes are involved in attachment and penetration. The proteins, named MICs, in these organelles are secreted, before the exocytosis of the rhoptry proteins. Before they get to their compartment, these proteins which have a classic hydrophobic amino terminal signal peptide move through the endoplasmic reticulum and exit the network at the golgi complex. They additionally have two conserved motifs at the C terminus - the first being a SYHYY and the second, an acidic series, EIEYE. These are important in folding, sorting and the spanning of the membrane. There are also distinct adhesive domains in the middle.
As soon as the tachyzoite attaches to the host cell, there is a marked increase in calcium levels in the parasitic cell, as calcium from the acidocalcisomes is released. This triggers the activation of at least two calcium dependant protein kinases that cause further signal cascades, and ultimately the release of micronemal proteins. The adhesive domains are involved in the adhesion and further invasion may take place.
Rhoptry proteins, reviewed in an article in Nature Reviews Microbiology,2008 , by Boothroyd, is conserved in its presence across the phylum and yet is varied in its architecture and numbers. There are 12 in the tachyzoite stages of Toxoplasma, each 2 to 3 micrometers in length. They appear to have compartments when studied with IFA. Protein compartmentalization post-transcriptionally occurs by understudied mechanisms. Rhoptries are even visible under a phase contrast microscope and appear to be related to exosomes. Of the 29 proteins identified in the rhoptries, 24 ROP proteins localize in the bulb region and 5 RON proteins localize in the neck region, with 28 newly recognized proteins yet to be allotted designations/names. Some of the ROP proteins are enzymes like kinases, proteases and phosphatases, that do not have orthologs in related genera. The, one gene- one protein, RON proteins however, have orthologs in related extant sps, although their exact functions are yet to be fully elucidated. The rhoptries also seem to possess lipids rich in cholesterol. These are suspected to play a role during invasion, by forming vesicles around secreted proteins and ultimately fusing with the parasitophorous vacuole (PV).
During cellular invasion, the rhoptry contents are released. Some of the RON proteins are involved in the formation of the moving junction, which forms the physical interface, that is , the point of contact during invasion. The ROP proteins, after their release, localize in the PV or the PV membrane (PVM) or inside the host cell (including the nucleus), with many other targets being yet unknown. ROP1 is released in the PV as a vesicle and later moves to the membrane. ROP2 and its associates are putative integral PVM proteins that recruit the host mitochondria to the cytoplasmic face of the PVM. PP2C-hn and ROP16 contain typical NLS (Nuclear Localization Signal) sequences that cause them to move to the nucleus. All these molecules seem to play a key role in the biology of Toxoplasma sps.
The exact mechanism of conservation of the apical complex across the phylum seems to be a curious thing, as the phylum seems to be inhabited by protozoans that are as different, from each other in their pathogenesis and habitat, as the night is from the day. It certainly is true that the use of the apical complex is not always the same in all species, and certainly not all have the above described mechanism of Toxoplasma gondii. Thus, the Toxoplasma model, despite being the best that we have got right now, gives us only a glimpse of the preeminence of the great phylum-defining feature. It surely is our starting point. Where we will yet reach from here is unknown . Will we ravel the marvels of the pathogenesis of, say Cyclospora, by studying this feature? Will we bring to light the reason for the bizzare behaviour of the other Apicomplexans ? We can only hope so, for now, because, we have miles to sail in our research before we reach the shores of understanding .
References :
1. Boothroyd JC, Dubremetz JF. Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat Rev Microbiol 2008;6:79-88.
2. Hu K, Roos DS, Murray JM. A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J Cell Biol 2002;156:1039-1050.
3. Soldati D, Dubremetz JF, Lebrun M. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int J Parasitol 2001;31:1293-1302.
Thursday, February 13, 2014
Oo-complexity - a note on the Eimeria oocyst
As is well known among poultry professionals, subclinical coccidiosis is a huge challenge to the industry. Reinfection in poultry houses is the single most important route of disease sustenance in the population, especially in the deep-litter system. Caged and raised cage systems, although much more effective in keeping other infections low, do not completely isolate the birds from infective stages. Excellent husbandry practices and prophylactic medication are the mainstays of infection-reduction plans. To say that current control strategies are not perfect is an understatement. All that can be done is being done and yet we are getting nowhere. Given the production parameters on high throughput farms, the sheer number of birds raised poses a big economic challenge. Cost of control adds to the variable production cost and raises the cost of production per unit of meat/eggs. The biggest problem with coccidiosis control lies in our inability to control of the oocysts of the parasitic genus, Eimeria.
Eimeria is an obligate protozoan parasite that belongs to the phylum Apicomplexa. The lifecycle is complex and monoxenous (sexual and asexual development takes place in the same definitive host). The oocysts that are formed at the end of sexual development are shed in the feces. They sporulate in the environment and are infectious to susceptible birds.
(Source : http://www.ars.usda.gov/Main/docs.htm?docid=11018; Creative Commons License)
Host and site specificity is maintained during infection. The variability in the virulence of the parasite allows it to cause either hemorrhagic enteritis or malabsorptive enteritis. Now, if you have ever taken a college course in Protozoology, you might already know all that. You might also know that appropriate temperature and oxygen conditions are needed for sporulation, at the end of which the infectious oocyst contain four sporocysts with two sporozoites each (Eimeria sps.). But, let us move in a little deeper, beyond textbook material. What makes the parasite tick (no pun intended)? How is such a complex lifecycle so effortlessly maintained?
The answer lies in the utter complexity of the oocyst .
(Source : http://www.ars.usda.gov/Main/docs.htm?docid=11018; Creative Commons License)
Epidemiologically, it has long been recognized that altering the transmission stage of a pathogen can adversely affect it's survivability. This lends credence to the idea that changing the temperature, pH , oxygen availability etc among other environmental factors changes oocyst viability and transmissibility.
The oocyst is quite resistant, to dessication and hence destruction. In terms of parasitic survival, the oocyst occupies a marvellous functional niche. It can be favorably compared to the eggs of birds with the oocyst wall performing the same function as the calcareous shell, providing strength , protection and stability.
Equivalent to hatching is the process of excystation, by which infective sporozoites are released at the predilection site. The oocyst wall is sturdy enough to resist extreme challenges like the action of detergents (to which most bacteria are susceptible), mechanical force, enzyme action and strong chemical reagents such as bleach and potassium dichromate. Sodium hypochlorite at various strengths removes parts of the oocyst wall.
In a recent study, Jenkins et al., compared the "Differing susceptibilities of Eimeria acervulina, E. maxima and E. tenella oocysts to dessication". The group concluded that Eimeria maxima oocysts displayed the highest resistance to dessication and were able to survive even in dry litter material. (Jenkins et al., 2013)
The protein rich oocyst wall starts out as electron dense wall forming bodies (WFB) in the endoplasmic reticulum and cytoplasm of the macrogamete. The veil forming bodies, WFB1 and 2 , are sequentially released after 'fertilization' of the macrogamete by the microgamete, to form the bilayered oocyst wall. [The layers are separated by a zone that spans 40 nm, (allowing the outer wall to be stripped away by bleach)] .
The wall forming bodies are chock full of proproteins that ultimately form the wall after post-translational modifications viz. N and C terminal modifications. Biochemical analysis of the wall proteins, using the combined gas chromatography- mass spectrometry technique, by Mai et al. revealed that the walls are mainly made up of proteins in which five amino acids predominate , viz. alanine , proline, valine, aspartic acid and isoleucine. Others have found that tyrosine also predominates and is important in dityrosine-protein cross linking. Lipids and carbohydrates contribute a little to the wall, with the predominant moieties being palmitic acid, stearic acid, oleic acid, linoleic acid, behenic acid, lignoceric acid, cholestane and cholesterol. (Mai et al., 2009)
So , what exactly confers the complexity ? It seems to me that the more we learn about the oocyst wall, the more we understand that there is more that we do not understand. Some questions remain. How do the dityrosine protein crosslinkings provide such enormous strength? What genes encode these proteins? Are there multiple genes that control these functions? Is the nature of the oocyst similar across the apicomplexans? Is it in anyway similar to egg walls of multicellular eukaryotic parasites? Comparative molecular parasitology might be the only way to answer these questions that deal with the very nature of the fabric of life of the oocyst.
References :
Eimeria is an obligate protozoan parasite that belongs to the phylum Apicomplexa. The lifecycle is complex and monoxenous (sexual and asexual development takes place in the same definitive host). The oocysts that are formed at the end of sexual development are shed in the feces. They sporulate in the environment and are infectious to susceptible birds.
Host and site specificity is maintained during infection. The variability in the virulence of the parasite allows it to cause either hemorrhagic enteritis or malabsorptive enteritis. Now, if you have ever taken a college course in Protozoology, you might already know all that. You might also know that appropriate temperature and oxygen conditions are needed for sporulation, at the end of which the infectious oocyst contain four sporocysts with two sporozoites each (Eimeria sps.). But, let us move in a little deeper, beyond textbook material. What makes the parasite tick (no pun intended)? How is such a complex lifecycle so effortlessly maintained?
The answer lies in the utter complexity of the oocyst .
Epidemiologically, it has long been recognized that altering the transmission stage of a pathogen can adversely affect it's survivability. This lends credence to the idea that changing the temperature, pH , oxygen availability etc among other environmental factors changes oocyst viability and transmissibility.
The oocyst is quite resistant, to dessication and hence destruction. In terms of parasitic survival, the oocyst occupies a marvellous functional niche. It can be favorably compared to the eggs of birds with the oocyst wall performing the same function as the calcareous shell, providing strength , protection and stability.
Equivalent to hatching is the process of excystation, by which infective sporozoites are released at the predilection site. The oocyst wall is sturdy enough to resist extreme challenges like the action of detergents (to which most bacteria are susceptible), mechanical force, enzyme action and strong chemical reagents such as bleach and potassium dichromate. Sodium hypochlorite at various strengths removes parts of the oocyst wall.
In a recent study, Jenkins et al., compared the "Differing susceptibilities of Eimeria acervulina, E. maxima and E. tenella oocysts to dessication". The group concluded that Eimeria maxima oocysts displayed the highest resistance to dessication and were able to survive even in dry litter material. (Jenkins et al., 2013)
The protein rich oocyst wall starts out as electron dense wall forming bodies (WFB) in the endoplasmic reticulum and cytoplasm of the macrogamete. The veil forming bodies, WFB1 and 2 , are sequentially released after 'fertilization' of the macrogamete by the microgamete, to form the bilayered oocyst wall. [The layers are separated by a zone that spans 40 nm, (allowing the outer wall to be stripped away by bleach)] .
The wall forming bodies are chock full of proproteins that ultimately form the wall after post-translational modifications viz. N and C terminal modifications. Biochemical analysis of the wall proteins, using the combined gas chromatography- mass spectrometry technique, by Mai et al. revealed that the walls are mainly made up of proteins in which five amino acids predominate , viz. alanine , proline, valine, aspartic acid and isoleucine. Others have found that tyrosine also predominates and is important in dityrosine-protein cross linking. Lipids and carbohydrates contribute a little to the wall, with the predominant moieties being palmitic acid, stearic acid, oleic acid, linoleic acid, behenic acid, lignoceric acid, cholestane and cholesterol. (Mai et al., 2009)
So , what exactly confers the complexity ? It seems to me that the more we learn about the oocyst wall, the more we understand that there is more that we do not understand. Some questions remain. How do the dityrosine protein crosslinkings provide such enormous strength? What genes encode these proteins? Are there multiple genes that control these functions? Is the nature of the oocyst similar across the apicomplexans? Is it in anyway similar to egg walls of multicellular eukaryotic parasites? Comparative molecular parasitology might be the only way to answer these questions that deal with the very nature of the fabric of life of the oocyst.
References :
1. Belli SI, Smith NC, Ferguson DJ. The coccidian oocyst: a tough nut to crack! Trends Parasitol 2006;22:416-423.
2. Jenkins MC, Parker C, O'Brien C, et al. Differing susceptibilities of Eimeria acervulina, Eimeria maxima, and Eimeria
tenella oocysts to desiccation. J Parasitol 2013;99:899-902.
3. Mai K, Sharman PA, Walker RA, et al. Oocyst wall formation and composition in coccidian parasites. Mem Inst
Oswaldo Cruz 2009;104:281-289.
Monday, January 6, 2014
An Apicomplexan by any other name ..... is still a Protozoan
Frank E. Cox, of the London School of Hygiene and Tropical Medicine, makes the most unflattering accusation in his article "Systemics of Parasitic Protozoa". He asserts that 'parasitologists' have completely ignored the relationships revealed by 'protozoologists', between and among non-parasitic protozoan groups and that they "continue to be embedded in the classifications of the 1980s" (Cox, 2002). Ooh, ouch ! I feel that such a great wrong must soon be corrected. So, let us get right to it !
The scientific community has moved on from Whittaker's Five Kingdom classification system that you and I learnt in school. Cavalier-Smith, in 1998, proposed a "Revised Six Kingdom System" in which all botanical life was classified into Kingdoms Fungi, Plantae and Chromista, zoological species into Protozoa and Animalia, while Bacteria were awarded their own kingdom (Cavalier-Smith,1998). In an earlier paper, he proposed 18 phyla under the Kingdom Protozoa, of which Phylum Apicomplexa was but one.
Other classification styles developed before this time, rule that the three phyla Apicomplexa, Dinozoa (Dinoflagellates) and Ciliophora (Ciliates like Paramecium) must be grouped together under the monophyletic group Alveolata. All members have a subsurface alveoli, microtubules, mitophores and mitochondria with ampulliform or tubular cristae. (Adl et al.,2005)
Cavalier-Smith proposed that the phylum Apicomplexa be further subdivided into two subphyla : Apicomonada and Gamontozon, the latter of which is divided into "infraphylum" Sporozoa (whose members have nine singelet centrioles, complete conoids and conidial rings and the general presence of oocysts and sporocysts in their life cycles) and infraphylum Hematozoa (whose members have a more primitive centriole and lack the presence of oocysts and sporocysts, instead undergoing merogony in vertebrate erythrocytes and gametogony in arthropod guts). (Cavalier-Smith,1993)
In his letter, Cox suggests that based on new analysis Apicomonada has lost its supposed relatedness to the other Apicomplexans (which certainly is true, because it had initially contained only mollusc parasites), and so Sporozoa must be restored to Phylum status (Cox, 2002). This suggestion was apparently not widely accepted, as proved by latter scientific articles that still call the phylum Apicomplexa. Textbooks including popular ones like 'Georgis' Parasitology for Veterinarians' get around this dilemma by adding 'Sporozoa' in parentheses after 'Apicomplexa'.
The traditional grouping of the Apicomplexans, on the basis of phenotype, host, tissue and vector, has been under four broad categories : Coccidians, Gregarines, Haemosporidians and Piroplasms.Each of the groups are defined below:
Coccidia : "host specific, intracellular parasites of the intestines and other organs, of vertebrates with alternating asexual and sexual phases of development resulting in the production of environmentally resistant oocysts in the feces of definitive hosts" (Barta , 2009)
Gregarines : " extra/intra cellular protozoan parasites with large mature gamonts that develop extracellularly with most exhibiting syzygy in their developmental cycles" (Barta , 2009)
Hemosporidians : "obligate heteroxenous blood parasites that undergo sexual development in Dipteran flies and asexual development in vertebrate host" (Dimitrov, 2013)
Piroplasms : Pleomorphic, heteroxenous with an incomplete apical complex; lacks an oocyst stage and flagella (Adl,2005)
Molecular phylogenetic analysis has only added to the confusion by suggesting corrections to canonical taxonomic nomenclature, that is more often not followed by others save the original authors of scientific papers. In the midst of this melee, Cryptosporidium occupies an unique phylogenetic niche. It does not exhibit the cellular vampirism exhibited by the dinoflagellates and gregarines, does not possess variant surface proteins and lacks an apicoplast (a genome- containing plastid-like organelle, homologous to chloroplasts). Analysis of the small subunit ribosomal RNA suggests that the genus is more closely related to the Archigregarines than the Coccidians, but still forms its own clade. With the Archigregarines, Cryptosporidium shares these important characters : monoxenous life cycle, oocyts with four sporozoites, a usual location in the host gastrointestinal tract and extracellular gamonts or trophozoites. (Barta , 2006)
The Tree of Life Web Project has the following "hypothetical tree" for the phylum Apicomplexa showing the major branches viz. clades:
(http://tolweb.org/onlinecontributors/app?service=external/ViewImageData&sp=46943 ; Creative Commons License)
At long last, we come to the real problem. The real question. If taxonomy is so inconstant, how will we/one study diversity and (dare I say it) evolution? There has been a clamour for a taxonomic scheme that will reflect phylogeny.
With this in mind, Morrison D. in a paper titled "Prospects for elucidating the phylogeny of Apicomplexa" lists five important changes that need to occur before any useful, directed progress can be made.
1. Taxon sampling
Anthropocentric research driven by economics and the relative veterinary-medical interest of the parasite has resulted in small, biased sample-data that do not adequately define the boundaries of taxa. Genebank is chock full of Plasmodium, Cryptosporidium, Theileria, Babesia and Toxoplasma sequences, whilst the Eimeria, Sarcocystis, Isospora and Gregarina have few to no representatives. Morrison also states that outgroups and basal taxa must be better studied to help classify already known species into clades.
The accusation of skewed sampling seems quite true. A search on NCBI revealed that as of today (6 Jan 2014), only 47 Apicomplexan species (of the many thousands that exist) have had their complete genome sequenced. All these are virulent parasites of animals/man.
2. Multiple molecular data sets
Frequently, trees are constructed on the basis of one gene (Quite simply because whole genomes are not available to play around with). Such trees, however, are not a true reflection of the relationship between species and clades. Among the Apicomplexans, the 18rRNA gene is most studied. But this gene is most prone to variation in terms of copy number. Other genes commonly studied are the HSP70, Actin, and a few mitochondrial genes. Morrison suggests that analyses be made with multiple gene sets, making sure that both nuclear and organellar genes (the latter show maternal inheritance) are included. Organellar genes can include mitochondrial genes and apicoplast (when they exist) genes.
3. Phylogentic analyses
Sequence alignment and tree building using the default parameters built into bioinformatic tools is naive, reproaches Morrison. Artifacts may arise from sequence length variation due to indel events, compositional variations like AT content and will not be resolved by multiple analyses (neighbor-joining, max-likelihood etc). Data must not violate the assumptions of the many analyses.
4. Reinterpretation of homologies
The thing about new knowledge is that it must agree or disagree completely or in degrees with old knowledge. Often reinterpretation is essential if we intend to get something worthwhile out of all our research efforts at all. A great example cited by Morrison, and which I spoke about in my previous post is the ineffectiveness of anti-coccidials on Cryptosporidium, easily explained by the genus not belonging to Coccidia.
5. Directed Data Collection
"The collection of pertinent data for the Apicomplexa can be best described as haphazard, which is unlikely to be of much practical value phylogenetically", laments Morrison. He proposes the formation of an informal group which would more likely be able to reach a consensus, over an autocratic formal group or a lackadaisical large group.(Morrison, 2008). I agree with him in that, for any progress from these mires of ignorance, directed data collection is essential.
Some, with adequate reason, have no interest at all in the systemics of taxonomy. Others are absolutely enthralled by the nuances of biological nomenclature. I, being one of the latter, have tried to understand the complexity of the system and have briefly presented the above the way I have understood it. Undoubtedly, the above is not absolute and is subject to change. But, change we will (our understanding and even our knowledge base) when the time comes. Till then, I'll leave you here on the shores of (the land of ) Systemics. So long. Farewell !
References :
1. Adl SM, Simpson AG, Farmer MA, et al. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 2005;52:399-451.
2. Barta JR, Thompson RC. What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends Parasitol 2006;22:463-468.
3. Cavalier-Smith T. Kingdom protozoa and its 18 phyla. Microbiol Rev 1993;57:953-994.
4. Cavalier-Smith T. A revised six-kingdom system of life. Biol Rev Camb Philos Soc1998;73:203-266.
5. Cox FE. Systematics of the parasitic Protozoa. Trends Parasitol 2002;18:108.
6. Moore RB, Oborník M, Janouskovec J, et al. A photosynthetic alveolate closely related to apicomplexan parasites. Nature 2008;451:959-963.
7. Dimitrov, D., Valkiunas, G., Zehtindjiev, P., Ilieva, M., & Bensch, S. (2013). Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacilla flava), with description of in vitro ookinetes of Haemoproteus motacillae. Zootaxa, 3666(3), 369–381.
8. Morrison DA. Prospects for elucidating the phylogeny of the Apicomplexa.Parasite. 2008;15(3):191-6.
9. Šlapeta, Jan and Victoria Morin-Adeline. 2011. Apicomplexa Levine 1970. Sporozoa Leucart 1879. Version 18 May 2011. http://tolweb.org/Apicomplexa/2446/2011.05.18 in The Tree of Life Web Project
The scientific community has moved on from Whittaker's Five Kingdom classification system that you and I learnt in school. Cavalier-Smith, in 1998, proposed a "Revised Six Kingdom System" in which all botanical life was classified into Kingdoms Fungi, Plantae and Chromista, zoological species into Protozoa and Animalia, while Bacteria were awarded their own kingdom (Cavalier-Smith,1998). In an earlier paper, he proposed 18 phyla under the Kingdom Protozoa, of which Phylum Apicomplexa was but one.
Other classification styles developed before this time, rule that the three phyla Apicomplexa, Dinozoa (Dinoflagellates) and Ciliophora (Ciliates like Paramecium) must be grouped together under the monophyletic group Alveolata. All members have a subsurface alveoli, microtubules, mitophores and mitochondria with ampulliform or tubular cristae. (Adl et al.,2005)
Cavalier-Smith proposed that the phylum Apicomplexa be further subdivided into two subphyla : Apicomonada and Gamontozon, the latter of which is divided into "infraphylum" Sporozoa (whose members have nine singelet centrioles, complete conoids and conidial rings and the general presence of oocysts and sporocysts in their life cycles) and infraphylum Hematozoa (whose members have a more primitive centriole and lack the presence of oocysts and sporocysts, instead undergoing merogony in vertebrate erythrocytes and gametogony in arthropod guts). (Cavalier-Smith,1993)
In his letter, Cox suggests that based on new analysis Apicomonada has lost its supposed relatedness to the other Apicomplexans (which certainly is true, because it had initially contained only mollusc parasites), and so Sporozoa must be restored to Phylum status (Cox, 2002). This suggestion was apparently not widely accepted, as proved by latter scientific articles that still call the phylum Apicomplexa. Textbooks including popular ones like 'Georgis' Parasitology for Veterinarians' get around this dilemma by adding 'Sporozoa' in parentheses after 'Apicomplexa'.
The traditional grouping of the Apicomplexans, on the basis of phenotype, host, tissue and vector, has been under four broad categories : Coccidians, Gregarines, Haemosporidians and Piroplasms.Each of the groups are defined below:
Coccidia : "host specific, intracellular parasites of the intestines and other organs, of vertebrates with alternating asexual and sexual phases of development resulting in the production of environmentally resistant oocysts in the feces of definitive hosts" (Barta , 2009)
Gregarines : " extra/intra cellular protozoan parasites with large mature gamonts that develop extracellularly with most exhibiting syzygy in their developmental cycles" (Barta , 2009)
Hemosporidians : "obligate heteroxenous blood parasites that undergo sexual development in Dipteran flies and asexual development in vertebrate host" (Dimitrov, 2013)
Piroplasms : Pleomorphic, heteroxenous with an incomplete apical complex; lacks an oocyst stage and flagella (Adl,2005)
Molecular phylogenetic analysis has only added to the confusion by suggesting corrections to canonical taxonomic nomenclature, that is more often not followed by others save the original authors of scientific papers. In the midst of this melee, Cryptosporidium occupies an unique phylogenetic niche. It does not exhibit the cellular vampirism exhibited by the dinoflagellates and gregarines, does not possess variant surface proteins and lacks an apicoplast (a genome- containing plastid-like organelle, homologous to chloroplasts). Analysis of the small subunit ribosomal RNA suggests that the genus is more closely related to the Archigregarines than the Coccidians, but still forms its own clade. With the Archigregarines, Cryptosporidium shares these important characters : monoxenous life cycle, oocyts with four sporozoites, a usual location in the host gastrointestinal tract and extracellular gamonts or trophozoites. (Barta , 2006)
The Tree of Life Web Project has the following "hypothetical tree" for the phylum Apicomplexa showing the major branches viz. clades:
At long last, we come to the real problem. The real question. If taxonomy is so inconstant, how will we/one study diversity and (dare I say it) evolution? There has been a clamour for a taxonomic scheme that will reflect phylogeny.
With this in mind, Morrison D. in a paper titled "Prospects for elucidating the phylogeny of Apicomplexa" lists five important changes that need to occur before any useful, directed progress can be made.
1. Taxon sampling
Anthropocentric research driven by economics and the relative veterinary-medical interest of the parasite has resulted in small, biased sample-data that do not adequately define the boundaries of taxa. Genebank is chock full of Plasmodium, Cryptosporidium, Theileria, Babesia and Toxoplasma sequences, whilst the Eimeria, Sarcocystis, Isospora and Gregarina have few to no representatives. Morrison also states that outgroups and basal taxa must be better studied to help classify already known species into clades.
The accusation of skewed sampling seems quite true. A search on NCBI revealed that as of today (6 Jan 2014), only 47 Apicomplexan species (of the many thousands that exist) have had their complete genome sequenced. All these are virulent parasites of animals/man.
2. Multiple molecular data sets
Frequently, trees are constructed on the basis of one gene (Quite simply because whole genomes are not available to play around with). Such trees, however, are not a true reflection of the relationship between species and clades. Among the Apicomplexans, the 18rRNA gene is most studied. But this gene is most prone to variation in terms of copy number. Other genes commonly studied are the HSP70, Actin, and a few mitochondrial genes. Morrison suggests that analyses be made with multiple gene sets, making sure that both nuclear and organellar genes (the latter show maternal inheritance) are included. Organellar genes can include mitochondrial genes and apicoplast (when they exist) genes.
3. Phylogentic analyses
Sequence alignment and tree building using the default parameters built into bioinformatic tools is naive, reproaches Morrison. Artifacts may arise from sequence length variation due to indel events, compositional variations like AT content and will not be resolved by multiple analyses (neighbor-joining, max-likelihood etc). Data must not violate the assumptions of the many analyses.
4. Reinterpretation of homologies
The thing about new knowledge is that it must agree or disagree completely or in degrees with old knowledge. Often reinterpretation is essential if we intend to get something worthwhile out of all our research efforts at all. A great example cited by Morrison, and which I spoke about in my previous post is the ineffectiveness of anti-coccidials on Cryptosporidium, easily explained by the genus not belonging to Coccidia.
5. Directed Data Collection
"The collection of pertinent data for the Apicomplexa can be best described as haphazard, which is unlikely to be of much practical value phylogenetically", laments Morrison. He proposes the formation of an informal group which would more likely be able to reach a consensus, over an autocratic formal group or a lackadaisical large group.(Morrison, 2008). I agree with him in that, for any progress from these mires of ignorance, directed data collection is essential.
Some, with adequate reason, have no interest at all in the systemics of taxonomy. Others are absolutely enthralled by the nuances of biological nomenclature. I, being one of the latter, have tried to understand the complexity of the system and have briefly presented the above the way I have understood it. Undoubtedly, the above is not absolute and is subject to change. But, change we will (our understanding and even our knowledge base) when the time comes. Till then, I'll leave you here on the shores of (the land of ) Systemics. So long. Farewell !
References :
1. Adl SM, Simpson AG, Farmer MA, et al. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 2005;52:399-451.
2. Barta JR, Thompson RC. What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends Parasitol 2006;22:463-468.
3. Cavalier-Smith T. Kingdom protozoa and its 18 phyla. Microbiol Rev 1993;57:953-994.
4. Cavalier-Smith T. A revised six-kingdom system of life. Biol Rev Camb Philos Soc1998;73:203-266.
5. Cox FE. Systematics of the parasitic Protozoa. Trends Parasitol 2002;18:108.
6. Moore RB, Oborník M, Janouskovec J, et al. A photosynthetic alveolate closely related to apicomplexan parasites. Nature 2008;451:959-963.
7. Dimitrov, D., Valkiunas, G., Zehtindjiev, P., Ilieva, M., & Bensch, S. (2013). Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacilla flava), with description of in vitro ookinetes of Haemoproteus motacillae. Zootaxa, 3666(3), 369–381.
8. Morrison DA. Prospects for elucidating the phylogeny of the Apicomplexa.Parasite. 2008;15(3):191-6.
9. Šlapeta, Jan and Victoria Morin-Adeline. 2011. Apicomplexa Levine 1970. Sporozoa Leucart 1879. Version 18 May 2011. http://tolweb.org/Apicomplexa/2446/2011.05.18 in The Tree of Life Web Project
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