Life Cycle

In the Insect Vector

The African trypanosomes reside almost exclusively in the bloodstream and are transmitted by the bite of the tse-tse fly which acquires the infection while taking a bloodmeal, and returns the trypanosome to a vertebrate host in its saliva when it takes another bloodmeal. Because  this mode of transmission is by inoculation during biting this group of trypanosomes are also referred to as saliva-type or “Salivarian”. (T. cruzi, on the other hand, is transmitted by fecal contamination and is referred to as a “Stercorarian”). The range of African trypanosomiasis is determined by the range of the vector. Interestingly, only newly hatched tse-tse flies are competent to transmit the disease. Glossina is in fact a poor vector in nature since less than 1% of the flies are infected.

The ingested form that is infectious for the fly is termed the short-stumpy bloodstream trypomastigote, which is a non-dividing form. Following ingestion, the bloodmeal is retained within the midgut, and the parasite differentiates into a procyclic form and divides by binary fission. After about two weeks some procyclics migrate from the midgut through the hemocoel eventually reaching the salivary glands. At this point they differentiate through an epimastigote stage into a metacyclic trypomastigote stage, which is a non-dividing form infectious for the mammalian host. Metacyclic trypomastigotes are found in the salivary glands ~ 20 days after the bloodmeal, and there are  ~ 40,000 trypomastigotes/bite, but it takes only 400 to initiate an infection.

Click here to see steaming videos of procyclic T. brucei.

 In the Mammalian Host

The metacyclic trypomastigotes replicate at the site of infection. There may be an immune response causing inflammation (trypanosomal chancre) at the site of the bite. From there the trypomastigotes move via the lymphatics to the lymph nodes and then to the bloodstream. In T. gambiense infection, swollen cervical (neck) lymph nodes are referred to as Winterbottom’s sign. Long- slender bloodstream trypomastigotes divide by binary fission in the bloodstream, generating, on occasion, short-stumpy forms to continue the cycle in the tse-tse fly. The long-slender trypomastigotes are not infectious for the fly.  

GLUCOSE METABOLISM IN AFRICAN TRYPANOSOMES

The metabolism of the procyclic trypanosomes in the fly midgut or in culture differs dramatically from that of the bloodstream forms in the mammalian host.

Vertebrate 

cristae              RQ*                 TCA cycle          CN sensitivity

Stumpy              +/-                  0.12                        -                            -

Slender              -                     0.10                        -                            -

 Fly

Procyclic             ++                  1.0                          +                           +

Epimastigote      +++                1.0                          +                           +

Metacyclic           +/-                 0.1                           -                            -

*RQ = respiratory quotient (CO₂ amount divided by O₂ amount)

What is the basis for the switch in metabolism? In the vertebrate the parasite uses the regulatory mechanisms of the host and utilizes the plentiful energy source of the blood, glucose. The segregation of glycolytic enzymes in the glycosome organelle substantially increases the efficiency of glycolysis. Oxygen is consumed via a plant-like alternative oxidase, which does not produce ATP by oxidative phosphorylation. The differentiation from the long slender to the short stumpy form in the bloodstream involves changes in metabolism. The stumpy forms are infective for the fly. In the fly glucose is limiting and therefore a more efficient utilization of glucose and amino acids occurs via the TCA acid cycle and oxidative phosphorylation. Metacyclics anticipate transfer to the vertebrate host by the mitochondrion by losing cristae and TCA cycle enzymes.

Differentiation of T. brucei

The differentiation from the stumpy bloodstream form into the procyclic form can be studied in culture and involves massive mitochondrial biogenesis. It provides a model system for the study of mitochondrial biogenesis in general.

From Mathews (1999).  Long-slender (A), short-stumpy (B) and procyclic forms (C) of the African trypanosome, Trypanosoma brucei. The basic biological characteristics of each cell type are shown to the right of each image. VSG, variable surface glycoprotein. Scale bars = 10 m.

The differentiation from long slender (LS) to short stumpy (SS) occurs in culture in so-called pleomorphic strains. It appears to be induced by cell density apparently through a low molecular weight factor in the medium. Long slenders are affected more by immune lysis than the short stumpies.

Also from Mathews (1999). Representation of the different phases of the course of a Trypanosoma brucei bloodstream parasitaemia. In Phase 1, the parasite population increases in number due to proliferation of slender-form parasites .Above a critical cell density, the slender-cell population initiates a phase of `differentiation-divisions', which generate stumpy forms (Phase 2). The stumpy forms do not divide and are competent for differentiation into the procyclic form, either when taken up in a tsetse bloodmeal, or in vitro. In Phase 3, the population is composed predominantly of intermediate and stumpy-form parasites; the population density eventually decreases as a result of antibody-mediated clearance of first slender cells and then stumpy cells (Phase 4). Finally, the parasite population is re-established by the outgrowth of slender-form parasites that have undergone antigenic switching (Phase 5).
 

The differentiation from SS to procyclic cells can also be studied in vitro. It is stimulated by the TCA  cycle intermediate, cisaconitate, and by lowering of the temperature to 27^oC. Other factors may also be involved in vivo such as glucose level and presence of proteases. The process can be monitored by assaying the gain of the insect cell -specific PARP cell surface protein and the loss of the bs-specific VSG coat.

AMINO ACIDS AND PROTEINS

Little is known about bloodstream forms. In T. gambiense the major amino acid utilized is alanine, with glucose yielding asp, glu, ala, gly. It also appears that bloodstream forms can take up proteins. Culture forms use proline as an energy source by reversal of the usual biosynthetic pathway to α- ketoglutarate. Presumably all insect stages utilize proline. Trypanosomes lack catalase/peroxidase hemoproteins as well as glutathione reductase. Thus they are sensitive to nitrofurans (Nifurtimox) which produce high levels of reactive oxygen intermediates—free radicals and hydroxyl radicals. Parasites contain a unique reductant called trypanothionine, which consists of two glutathione peptides conjugated to spermidine. Reduction is via trypanothionine reductase, an NADPH dependent reaction.

     Glutathione (glutamyl-cysteinyl-glycine)  + spermidine    = trypanothionine

Substrate specificity min-1 M-1     GSH reductase               TSH reductase

 Glutathione                                          1.8 x 108                      0.8 x 108

 Trypanothionine                                   1.4 x 108                      5.0 x 108

Melarsoprol inhibits trypanothionine reductase

Questions:

1. What is the difference between Salivarian and Stercorarian trypanosomes?

2. In the tsetse, the parasite undergoes a series of developmental changes. What are these changes and where do they occur?

3. In the mammalian bloodstream, the parasites also undergo a developmental change. What is the change and how is it stimulated?

4. What is the major difference in metabolism between the bloodstream forms and the procyclic forms?

5. What is the alternate oxidase?