PORTUGUESE

 PARASITOLOGY  -   CHAPTER THREE

 MOLECULAR PARASITOLOGY :  TRYPANOSOMES

 EUCARYOTIC CELLS WITH A DIFFERENT WAY OF DOING THINGS  

Dr Richard Hunt
Professor Emeritus
University of South Carolina School of Medicine
 

Trypanosome trypomastigotes (Trypanosoma spp.).
  © Dennis Kunkel Microscopy, Inc.  Used with permission

In this section, we shall look at the disease of trypanosomiasis and the life cycle of the parasites that cause it. The novel aspects of the life cycle lead us to ways in which the parasite has gained unusual biochemical pathways to cope with its niche. These offer potential sites for chemotherapy.

Trypanosomes are successful parasites which manage to escape the host's immune response; this happens by a very complex mechanism of antigen switching and it is the knowledge of this mechanism that has led us to the first steps in developing an anti-trypanosome vaccine.

TRYPANOSOMES

Trypanosomes belong to the order KINETOPLASTIDA, so-called because of the large DNA-containing structure, the kinetoplast, found at the base of the flagellum.

Figure 1. Tsetse fly: the vector for African trypansomiasis © Ohio State University, College of Biological Sciences

Figure 2 Winterbottom's sign (CDC from Parasites on the web)

The vector for African trypanosomiasis is theTsetse fly  (figure 1) and the distribution of the disease parallels the distribution of the vector (figure 4).

The symptoms of African trypansomiasis depend on host and the sub-species of trypanosome. In T. gambiense infections there is massive stimulation of immune system and complement-mediated lysis of host cells (gives characteristic anemia). Generalized pain, weakness, cramps and swelling of neck lymph nodes (Winterbottom=s sign, figure 2). Parasites invade all organs of the body including heart and CNS. The latter leads to apathy, mental dullness, tremors, convulsions and sleepiness, coma. There is rapid weight loss and death a few months later from malnutrition, heart failure, pneumonia or a parasitic infection. In the case of T. brucei rhodesiense infections, there is no coma or nervous system symptoms as probably patient dies before these can develop.

Recently on the increase, there are a minimum of 20,000 new cases a year; 50,000,000 people are at risk. Nagana prohibits cattle raising in a large area of Africa causing further malnutrition.

Structure of an African Trypanosome

Trypanosomes are unicellular protozoans (figure 3) with a single flagellum that contains microtubules in the 9+2 arrangement typical of other flagella. At the base of the flagellum is the kinetoplast (figure 3) which contains DNA in the form of about 6000 catenated circles. The kinetoplast DNA is 10% of the total cellular DNA and is the important site of action of some anti-trypanosome drugs such as ethidium. The kinetoplast is part of the single long mitochondrion which changes morphology during various stages of life cycle.

Most other organelles are those typical of any eucaryotic cell. At  surface of the cell are sub-membranous pellicular microtubules which give the trypanosome its shape. These underlie a typical  plasma membrane which is often covered by an electron-dense surface coat (figure 3).

Figure 3   

Epimastigotes grown in culture; kinetoplast (KP) is anterior to the nucleus (N). In most species of Trypanosoma, this stage reproduces in the gut of the vector. © Ohio State University, College of Biological Sciences

Trypomastigotes in blood smear; kinetoplast is posterior to the nucleus. This stage is found in all species of Trypanosoma, and in most species it is the only stage that reproduces in the vertebrate (human) host. © Ohio State University, College of Biological Sciences

  Thin section of a trypanosome (Leptomonas collosoma) showing the kinetoplast (k) which is the DNA-containing region of the single mitochondrion (m). The kinetoplast is found at the base of the flagellum and flagellar pocket (fp)

Figure 4    

   Diagram to show principal structures revealed by the electron microscope in the bloodstream trypomastigote form of the salivarian trypanosome, Trypanosoma congolense. It is shown cut in sagittal sections, except for most of the shaft of the flagellum and the anterior extremity of the body. (Adapted from Vickerman, K., 1969. J Protozool. 16:54-69.) (Note: the three sub-species (rhodesiense, gambiense and brucei) cannot be distinguished morphologically) 

 Life cycle of Trypanosoma rhodesiense  

 Distribution of West African or Gambian Sleeping Sickness and East African or Rhodesian Sleeping Sickness  

The distribution of tsetse fly  and cattle raising areas
 

Figure 5    

  During a blood meal on the mammalian host, an infected tsetse fly (genus Glossina) injects metacyclic trypomastigotes into skin tissue.  The parasites enter the lymphatic system and pass into the bloodstream .  Inside the host, they transform into bloodstream trypomastigotes , are carried to other sites throughout the body, reach other blood fluids (e.g., lymph, spinal fluid), and continue the replication by binary fission .  The entire life cycle of African Trypanosomes is represented by extracellular stages.  The tsetse fly becomes infected with bloodstream trypomastigotes when taking a blood meal on an infected mammalian host (, ).  In the fly’s midgut, the parasites transform into procyclic trypomastigotes, multiply by binary fission , leave the midgut, and transform into epimastigotes .  The epimastigotes reach the fly’s salivary glands and continue multiplication by binary fission .  The cycle in the fly takes approximately 3 weeks.  Humans are the main reservoir for Trypanosoma brucei gambiense, but this species can also be found in animals.  Wild game animals are the main reservoir of T. b. rhodesiense.

DPDx, Division of Parasitic Diseases CDC.  
 

Forms of T. Brucei in the mammalian and insect hosts

The various stages of the life cycle of T. brucei in each of its hosts can be distinguished morphologically (figure 5)

Biochemistry and molecular biology of African trypanosomes

Oxidative metabolism

All living organisms make ATP as an energy carrier. This is produced mainly by the oxidation of carbohydrates using glycolysis and the tricarboxylic acid (TCA) cycle (figure 6). Because free living organisms (like us) do not have an abundance of food, we rely on the much more efficient TCA cycle for most of our ATP production.

The trypanosome meets very different environments at different stages of its life cycle. In the mammalian blood stream there is an abundance of oxygen and glucose. The opposite is true in the insect gut or hemolymph. This is reflected in the number of reactions of glycolysis and TCA cycle that can be carried out and in the elaboration of the kinetoplast/mitochondrion. Clearly, if an organism can dispense with the TCA cycle it can also dispense with its mitochondrion.

The forms of T. brucei in the insect gut have a full complement of TCA and glycolysis enzymes. This is not surprising since nutrients are NOT abundant; in most organisms that use oxidative phosphorylation, ATP production is sensitive to cyanide because cytochromes a/a₃ react with cyanide and can no longer transfer an electron to oxygen to form water; however, oxidative phosphorylation in the insect gut forms of Trypanosomes is only PARTIALLY CYANIDE SENSITIVE. Cytochrome a/a₃ system is still CN^--sensitive but in Trypanosomes there is an additional cytochrome O which is CN^- - insensitive. Little is known about the cytochrome O system. In the insect, partial aerobic fermentation produces succinate, pyruvate, acetate as well as carbon dioxide. Proline is a major fuel source.

The forms of T. brucei in the mammalian bloodstream use only inefficient glycolysis because there is so much nutrient available; since glycolysis produces much less ATP than the TCA cycle, respiration is 50 times that of normal mammalian cell and in the bloodstream, T. brucei uses 10 times the amount of fuel as in insect gut.

Note from biochemistry lectures. If you use only glycolysis to make ATP as is done in anaerobic respiration in our muscles or in yeast, you cannot just excrete pyruvate, which is what the trypanosome does in fact do, since you will quickly reduce all of your NAD⁺ to NADH and the whole pathway will stop for lack of oxidized substrate. We convert pyruvate to lactate while yeast converts it to ethanol to oxidize our NADH back to NAD⁺ and keep glycolysis going. Trypanosomes excrete pyruvate and have another way of converting NADH back to NAD⁺.

In trypanosomes, dihydroxyacetone phosphate metabolism is necessary for reoxidation of NADH. This is an aerobic system, however, requiring oxygen but oxygen consumption is CN^- - insensitive so it does not use the usual cytochrome chain. An FAD-containing dehydrogenase linked to copper containing oxidase. This complex is the glycerophosphate oxidase system. Trypanosomes in the bloodstream depend on this mechanism of keeping NAD oxidized. This system is the target of two trypanocidal drugs: SURAMIN and SHAM (salicylhydroxamic acid) which is a chelating agent. Terminal oxidase contains copper. These drugs have their specificity because this is a metabolic pathway that mammals do not use.

There is another oddity in the trypanosomes' metabolism of carbohydrates. Unlike mammalian cell, first nine reactions of glycolysis are organelle-associated (GLYCOSOME). Why trypanosomes need to compartmentalize glycolysis is not clear. A compartment does mean that all of the enzymes are concentrated in one place but diffusion does not seem to be limiting in cells that do not have glycosomes.

Kinetoplast morphology

The morphology of the kinetoplast changes with metabolism carried out by the organism:

• Bloodstream slender trypomastigoes: simple mitochondrion, few cristae are short and tubular

• Bloodstream short-stumpy: elaboration of mitochondrion, synthesis of mitochondrial enzymes

• Fly midgut: elaborate array of plate-like cristae. Mitochondrion extends both anteriorally and posteriorally from kinetoplast

• Fly metacyclic: degeneration of mitochondrion 

  Figure 7 Electron micrograph of the surface coat of the mammalian form of a trypanosome. The pink arrow indicates the coat and the blue arrow indicates the underlying plasma membrane

  Figure 8 Successive waves of parasites in the blood are characteristic of sleeping sickness and correlate with cycles of fever.  A population of parasites (with a few variable surface glycoproteins (VSGs)) divides in the bloodstream over a period of days. Some of these trypanosomes have VSG A on their surface (clone A). The immune system raises antibodies against  all of the population's antigens and, as a result, most of the parasites die. A few trypanosomes, however, change their coat so that they express  another VSG (e.g. VSV B). These parasites survive by expressing the new VSG gene and  give rise to a new population (clone B). In time, the host raises antibodies against VSG B and clone B cells die off but again a few cells change their coats and survive.  This cycle is repeated many times in the course of a chronic infection as parasites keep expressing new genes and displaying new VSG antigens. From each successive population it is possible to isolate individual trypanosomes and from them to grow clones expressing particular VSGs.

Host reactions to the parasite

Any parasite faces a problem when it takes up residence within another organism. The latter is likely to mount an immune response to the parasite which may destroy it. Some parasites hide behind surfaces that disguise them so that to the immune system they look like normal cells. Other parasites (including Trypansoma cruzi) enter cells to get away from the immune system. T. brucei does neither. Instead,

The parasite is very antigenic which is one reason for the symptoms shown by the infected patient. But the number of parasites in the bloodstream does not go on increasing and increasing until the patient dies. The patient undergoes waves of fever and cycles in parasite infestation. The waves of parasitemia correlate with the fever observed. Number of parasites in blood shows waves as the immune system partially overcomes the infection. Cyclic nature of parasitemia is very characteristic.

So why does not the massive immune response mounted by the infected individual clear the body of the parasite as it ought to do? Although massive immune response with strikingly high levels of Ig (especially IgM) and profound B lymphocyte proliferation, it must be that there is a change effected in some trypanosomes of the total population that allows them to seed a new generation of parasites. Later in the chronic phase of the infection, lymphoid organs are depleted of lymphocytes, they shrink and patchy fibrosis replaces the lymphocytes. Immunodepression sets in and the parasitemia is uncontrolled leading to death.

Why are not all of the parasite destroyed by the massive immune response? This is the key to the progress of the disease! The answer must lie in the electron-dense surface coat (figure 7) that surrounds the bloodstream forms of the parasite and which is the only major antigen recognized by the host=s immune system.

If we clone single cells from different infected animals or patients, the coat is biochemically different! Not just a bit different but so different that the coat protein must come from the expression of different genes by trypanosomes in each animal. Moreover, if we take cells from a defined wave of parasitemia in the same patient, it is found that all of the trypanosomes in that wave of organisms are expressing the same single surface antigen whereas in other waves, all of the parasites are expressing a single but completely different antigen (figure 8). In other words, a different surface antigen gene is being expressed. The surface coast is therefore made of VARIABLE SURFACE ANTIGENS or VARIABLE SURFACE GLYCOPROTEINS (VSGs). You will also see the term VATs for variable antigen types.

Thus escape from the immune response depends upon the ability to express a new VSG. Since hundreds of these waves of parasitemia can occur before the host dies (in a laboratory situation, normal number of waves is much fewer that this) and no antigen is repeated, there must be an equal number of VSG genes. In fact, there are probably 1000-2000 such genes C10% of the cell=s genome is devoted to genes that express these surface molecules that allow the organism to be one step ahead of the host's immune response.

This leads to a series of questions: What is the structure of these VSGs? How is the switch effected? How is only one VSG gene is expressed at a time? How is complete release of the VSG carried out?

Each VSG glycoprotein has a size of about 65kD, about 500 amino acids and has three domains. At the N-terminus is the signal sequence; the next 360 amino acids are usually very different from the similar sequence in other VSGs. The 120 C-terminal amino acids are quite similar in different VSGs. This latter part is hidden from the immune system by being next to the plasma membrane.

From protein sequencing and cDNA sequencing we get a different picture of the C-terminal part of the molecule. The cDNA (gives sequence encoded by gene) shows a typical transmembrane hydrophobic sequence that is used normally to attach the protein to the plasma membrane together with a short intracellular domain but protein sequencing shows that the transmembrane part of the protein and the intracellular part are no there in the mature protein. They are replaced by a weird structure that contains sugars, ethanolamine, phospho-inositol and fatty acids. This structure is common to all VSGs and is highly antigenic when purified but not in vivo. This suggests that the VSGs in the coat are tightly packed to exclude antibodies. Therefore this site is of nor use in vaccine development.

How does the sequential expression of VSGs occur?

Restriction endonuclease digestion, gel electrophoresis and Southern blotting allows us to fragment the trypanosomes= genome and separate the fragments. We can now detect a particular gene in the genome displayed on our gel by hybridizing it with a complementary probe. This is usually cDNA made against mRNA or against a cloned gene.

If we are lucky with our fragmentation and there is only one gene in the genome for a particular protein, we should see only one band that will hybridize on the gel (providing that an endonuclease has not cleaved within the gene). Such analyses showed that each VSG gene was normally expressed only once  in the genome which is not surprising since VSG genes constitute so much of the genome and multiple copies would occupy a very large proportion of the genome.

We can take DNA from organisms in the first, second, third and fourth waves of  parasitemia (clones A, B, C and D). We can then probe these DNAs after fragmentation with a probe that detects the gene expressed in second wave, that is in clone B. We find that clones A, C and D (which are not expressing VSG B) have only one copy of the VSG B.

However, a very surprising result was found, as shown in the diagram at the left (figure 9), when we probe for the gene that codes for VSG B in cells that are expressing VSG B.

There is an extra copy of the gene that is being expressed in this particular clone of trypanosomes. IT IS ONLY THE GENE THAT IS BEING EXPRESSED THAT OCCURS IN AN EXTRA COPY! THUS WE HAVE GENE DUPLICATION but it is only temporary as when the trypanosome switches to a new VSG the extra copy usually disappears and is replaced by another EXPRESSION-LINKED COPY (ELC). It is the extra copy that is being transcribed into mRNA for translation into protein, not the copy that is permanently in genome. The new copy is in an EXPRESSION SITE. Expression sites are always close to a TELOMERE, that is near the end of a chromosome. An ELC and a permanent gene may be on different chromosome. This suggests a copy/translocate mechanism for a cassette of information. Sometimes, an expression linked copy is not produced bu the permanent copy is transcribed. These transcribed non-ELC genes are always at telomeres. The temporary copying of a gene and using the copy is very unusual.

Can the genes be expressed in any order? Is there a preprogrammed sequence of expression? One would suspect not since there are thousands of VSG genes and there would be a selective advantage of being able to use all of them. Indeed, the order is not absolute. At the beginning of the infective phase, VSGs are produced by parasites in the insect salivary glands. A subset of the repertoire (about 12) of the VSGs are produced here. This remains during the first wave of parasitemia in the mammal. The whole repertoire is then open to expression and there is preferred but not fixed order of expression. Each gene can be expressed only once during an infection. Note that the fact that there is an initial subset of VSGs that are expressed gives hope for a vaccine.

mRNAs in trypanosome are also very unusual. Almost every mRNA that has been looked at starts with the same 35 nucleotides. This is coded by an exon far away from the rest of the gene.

Shedding of the VSG coat

It will clearly be important that the parasite sheds all of its coat at a given time in order to survive the developing immune response against its original VSG. We saw earlier that the form of the VSG at the cell surface is attached through a glycolipid and not via a transmembrane protein sequence. This may give a clue to how shedding occurs. A trypanosome- and VSG-specific phospholipase C is found in bloodstream forms. Thus since all VSGs have the same attachment structure only one enzyme is needed for rapid and complete cleavage.

Possibilities for chemotherapy

The existence of possibly 2000 VSG genes makes a wide spectrum vaccine very unlikely. Unfortunately, the common antigens are buried away from the immune response. Perhaps an agent interacting with the 35 nucleotide sequence of the mRNAs that are found in all trypanosomes but in no host animals mRNAs could be effective but none is known. The best possibility is a vaccine against the 12 or so initial antigens that are expressed in the organisms that are injected by the tsetse fly. Alternatively, an inhibitor of the phospholipase C might be a very good candidate.

Extraordinary RNA editing by trypanosomes: The mystery of the missing genes

These organisms have proved to be biochemical oddities in many respects but when some of the findings in trypanosomes have been looked for in other species, these apparently unique biochemical processes have turned out not to be unique after all.

The trypanosomes have a very odd nuclear genome. The chromosomes do not condense in nuclear division and so we do not know how many chromosomes there are. The nuclear genes include 1000-2000 genes that encode the variable surface antigens that allow the coast of the organisms to be changed regularly so that it can avoid the host=s immune response. The way in which this is done is extraordinary and involves the shifting of a new copy of a gene into an expression site when it is needed. Up to 10% of the genome is composed of all of these genes for variable surface antigens.

In addition, there is the kinetoplast. This DNA-rich structure lies at the base of the flagellum and is at one end of the single long mitochondrion of the flagellate. It is equivalent to the mitochondrial DNA of all other cells but makes up a very much greater proportion of the DNA of the cell than does the single circle mitochondrial DNA of other cells. Remember that our mitochondria can code for a few of their own proteins (some cytochrome subunits and ribosome subunits) together with all of the mitochondrial ribosomal RNAs and all of the mitochondrial transfer RNAs. Although the kinetoplast DNA is much more elaborate and is a greater proportion of the cell's DNA, it does not code for any more RNAs or proteins than other mitochondrial DNAs. Indeed, some of the tRNAs of the kinetoplast are not encoded in this DNA and have to be imported from the cytoplasm which is not the case with mammalian mitochondria. The mechanism for this import is unknown.

The reason that kinetoplast DNA makes up such a high proportion of the total DNA of the cells is it complexity.

It contains 20-50 copies of a 22kb MAXI CIRCLE that is equivalent to mitochondrial DNA in any other mitochondrion. BUT in addition, there are up to 10,0000 1kb MINI-CIRCLES of , until recently, unknown function. These are odd in another way: they form a single network of catenated circles.

As already noted, among the things coded for by the maxi circle DNA are ribosomal RNA of mitochondrion and a variety of the enzymes of the mitochondria respiratory chain. There are also several unidentified open reading frames in the maxicircle DNA.

The enigma of the cytochrome oxidase of some trypanosomes, especially Trypansosma brucei

In all of the trypanosomes that have been looked at, the cytochrome oxidase subunit III (CO III) is encoded in the kinetoplast DNA. All, that is, except T. brucei. Not only do all but T. brucei have their COIII genes in the kinetoplast DNA, but they are all in the same position within that DNA. Yet with the exception of the COIII gene, T. brucei kDNA looks very like that of two other trypanosomes, Crithidia fasciculata and Leishmania tarentolae.

The only real difference is the lack of COIII gene. In the other two species, this gene is upstream of the apocytochrome b gene. Does this mean that T. brucei has no cytochrome oxidase subunit III? No it must have for it has to carry out oxidative phosphorylation. In addition, the mRNA and the protein are clearly in the organism. Perhaps the gene is just not in the same place as the similar gene in the other trypanosomes. But we cannot find it elsewhere. Probing the DNA either of the nucleus or the kinetoplast shows that there is no gene. How can we have a protein and an mRNA without a gene to code for it?

Since in other trypanosomes that clearly have the COIII gene, it is always in the same place, perhaps we should look again at this region of the kinetoplast genome! So what is there in this region of kDNA in T. brucei? There are a few open reading frames that might code for small proteins but nothing of the size of the COIII protein and, in any case, the two largest have no start AUG codon so cannot code for a real protein. Even if they did, they would code for a very unusual protein which would be very rich in charged amino acids for the DNA in this region is very G-C rich.

As already noted, investigators have looked for the COIII gene elsewhere in the kDNA maxi circle but not found it. The kDNA maxi and mini circles have now been sequenced. Using probes that will detect the COIII gene in L. tarentolae and C. fasciculata, we do not find any apparent COIII gene in maxicircle or the nuclear DNA of T. brucei.

This means that the COIII gene is either missing OR highly diverged so that probes do not pick it up. But, again as noted above, it cannot be missing as the T. brucei has a cyanide-sensitive cytochrome system similar to the other two. It would also not be expected to be highly diverged in view of the extremely high conservation of the COIII genes in all other trypanosomes that have been looked at.

If you cannot find the gene, one can look for the transcripts, i.e. the mRNAs.

Evidence was found for an mRNA transcript in T. brucei that had a sequence similar to the gene for COIII in the other trypanosomes. The similarity of the sequences allows the determination of the correct open reading frame for the T. brucei transcript.

Of the 181 amino acids predicted by the sequence, 135 conserved amongst all three. If we take conservative replacements and those conserved in one other species we find 160 out of 181 are conserved. This level of conservation (88%) is slightly better than that between T. brucei and L. tarentolae maxi circles genes where the conservation ranges from 65% to 84%.

This means that there must be a gene for the COIII protein in T. brucei but where is it?

Again we must ask: Is there a genomic sequence that matches the COIII transcript?

Before it has been shown that heterologous hybridization using L. tarentolae and C. fasciculata COIII probes could not detect COIII sequences in T. brucei genes. This was confirmed by Southern blot analyses using probes that were predicted form the sequence that was obtained for the transcript. The probes do not hybridize to kDNA or to total DNA.

No detection could be found with probes that would have detected 0.5 copies of the gene per genome.

Since there is no gene apparently for these transcripts in T. brucei, two possibilities:

1. The transcript may be made by splicing together small fragments of RNA transcribed form multiple sites. There is after all precedent for mini-exons in the trypanosome system. There are data that would tend to exclude this possibility.

2. There is severe editing of the transcript after or during transcription sot that the final transcript is nothing like the gene from which it was transcribed.

So now we must go back to the kinetoplast genome. The gene MUST be there, we just cannot detect it

and the place to look for the gene for COIII would be immediately upstream of the apo-cytochrome b gene where the COIII gene is located in so many other trypanosomes.

The sequence in this position which we have seen does not contain large open reading frames with start codons was reinvestigated. But, in fact, the sequence of this region matches the transcript sequence exactly EXCEPT for the presence of uridines in the transcript that are not in the DNA of the gene. Note: start codons are ATG so that explains the lack of start codons in the gene. The lack of 25% of the nucleotides explains the smaller open reading frames. This also explains the high G-C content.

In spite of the differences between the RNA and DNA sequences, only the number and positions of Us are affected; Cs, Gs, and As occur in the same sequence in the mRNA and the genomic DNA.

Of the 626 nucleotides sequenced initially in the T. brucei COIII transcripts, 347 are URIDINES that are not coded for by the gene. They are added in 121 different sites.

At one of those sites, the addition of a U creates a stop codon exactly where the native stop codon occurs in the other COIII genes.

In addition, 16 uridines, predicted by the genomic sequence at 7 sites appear to be deleted.

The protein coding portion of the transcript contains 315 additions and 15 deletions in 546 nucleotides.

Thus 58% of the coding part of the mRNA results from editing and is not in the original gene.

The editing is the reason why cDNA probes do no hybridize to the genomic DNA.

Why do this?

One suggestion for this editing is that it might serve as a control mechanism during the complex lifecycle of the parasite. Of course, not using Us in the gene saves space--or does it?

How does the editing take place? What determines where the Us are put in and taken out? Is there an original transcript that corresponds to the kinetoplast genomic sequence?

The first step is indeed to make a transcript that is complementary to the genomic sequence. This is now edited by putting in or taking out uridines at specific places. The sequence of a nucleic acid is usually determined by another nucleic acid and a whole new class of RNAs that are at present unique to the trypanosomes are used for this. These small RNAs are called guide RNAs (gRNAs) are coded for by regions of the maxi-circle and of the mini-circle for which previously no function was known.

One gRNA has a 5' end that is complementary to a short region of the unedited COIII initial mRNA transcript (near the 3' end). At the end of this region of the gRNA there will be one or more adenosines that are not in the primary transcript followed by more sequence and a poly U tail. At the site of non-complementarity the primary transcript is cut by an endonuclease and a U (from the poly U tail) is transferred on (by terminal U transferase). This is now complementary to the A in the gRNA. If there is another A in the gRNA, another U will be added to the primary transcript and this will go on till there is not another A in the sequence. This is the signal for a ligase enzyme to attach the original 5' end of the primary transcript to the newly inserted U (or Us) on the 5' end of the 3' portion of the primary transcript. Presumably now the gRNA dissociates. But now a new sequence containing Us is present in the initially edited primary transcript and this is recognized by a different gRNA which can edit further towards the 5' end of the primary transcript. Thus there is sequential editing from the 3' to the 5' end of the original mRNA transcript and intermediates in processing have been found that support this idea. Whatever, the mechanism, we have an extraordinary situation in which most of the uridines of a gene are left out and are put in after transcription to mRNA using the coding potential of another set of genes that code of the gRNAs. This seems very inefficient and has only been found in trypanosomes to this extent.

Trypanosoma cruzi

Chagas Disease

The disease is carried by reduvid bugs including the assasin bugs and rhodnius (figure 10) which infect the patient when they defecate after taking a blood meal

The symptoms of Chagas' disease are: chronic infection, neurological disorders (including dementia), megacolon (figure 11), megaesophagus, and damage to the heart muscle (figure 11). Chagas' disease is often fatal unless treated.

In acute disease, there is often severe anemia, muscle pain and neurological disorders. The latter are common in children under 2 years in which death may occur in about a month. Chronic disease may be mild and sometimes asymptomatic but there may be damage to nerves causing cessation of gut muscle contractions, irregular heartbeat and destruction of nervous system motor centers. The chronic form of the disease is found in adults but most likely arises from a childhood infection. T. cruzi can cross the placenta and so chronically-infected mothers can infect their babies which may succumb to the very acute form of the disease 

Figure 11      

Worldwide distribution of Chaga's disease

Amastigotes (pseudocyst) of T. cruzi in the heart of a dog. © Ohio State University, College of Biological Sciences
 

Trypanosoma cruzi in monkey heart.  CDC/Dr. L.L. Moore, Jr. 1969  

Megacolon in a Chagas' disease patient

WEB RESOURCES

Division of Parasitic Diseases - Centers for Disease Control

Like, the African trypanosomes, T. cruzi escapes from host immune response but it does not do so by changing its antigenic coat. Thus, there is no antigenic variation. Instead, it escapes by hiding inside cells. The disease starts after a bite by the insect vector. After 7-14 days the trypanosomes arrive in the lymph nodes where they divide. Here, they form aggregates called pseudocysts.. When the pseudocysts rupture, the released parasites can enter cells in various parts of the body including lymphatic tissue, muscle and tissue around nerve ganglia. The invasion of the cardiac nerve ganglia is the cause of much of the heart disease in areas where T. Cruzi is found.

 

Binding to and infection of the host cell

Binding

Amastigotes bind to the cell surface (e.g. a monocyte) via a variety of receptor proteins including fibronectin. Sialic acid is important as cells deficient in sialic acid are not penetrated. Interestingly, T. cruzi has an enzyme called trans-sialidase which actually puts more sialic acid onto cells, thereby enhancing uptake.

Entry into the cell 

Uptake is by a process of induced phagocytosis. Lysosomes migrate to the cell surface when cells come in contact with T. cruzi and the  parasite enters  a cytoplasmic vacuole, the parasitophorous vacuole, that is formed from a lysosome. Thus, agents that induce the migration of lysosomes from peri-nuclear areas so that they underlie the plasma membrane, enhance infectivity. Conversely, blocking lysosomal function stops infection. Somehow, the parasites escape from the destructive potential of the lysosome and after about an hour T. cruzi releases a protein toxin that inserts into the membrane at the low pH typical of this organelle. As a result T. cruzi  escapes into the cytoplasm as the lysosomal membrane is destroyed. 

  An infected triatomine insect vector (or “kissing” bug) takes a blood meal and releases trypomastigotes in its feces near the site of the bite wound.  Trypomastigotes enter the host through the wound or through intact mucosal membranes, such as the conjunctiva .  Common triatomine vector species for trypanosomiasis belong to the genera Triatoma, Rhodinius, and Panstrongylus.  Inside the host, the trypomastigotes invade cells, where they differentiate into intracellular amastigotes .  The amastigotes multiply by binary fission and differentiate into trypomastigotes, and then are released into the circulation as bloodstream trypomastigotes .  Trypomastigotes infect cells from a variety of tissues and transform into intracellular amastigotes in new infection sites.  Clinical manifestations can result from this infective cycle.  The bloodstream trypomastigotes do not replicate (different from the African trypanosomes).  Replication resumes only when the parasites enter another cell or are ingested by another vector.  The “kissing” bug becomes infected by feeding on human or animal blood that contains circulating parasites .  The ingested trypomastigotes transform into epimastigotes in the vector’s midgut .  The parasites multiply and differentiate in the midgut and differentiate into infective metacyclic trypomastigotes in the hindgut .
Trypanosoma cruzi can also be transmitted through blood transfusions, organ transplantation, transplacentally, and in laboratory accidents. 

 DPDx, Division of Parasitic Diseases CDC.  
 

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