Leishmania donovani – Evasion of the Host Immune Response

by KH

The human immune system is extremely complex and has a repertoire of immune cells with many functions.  However, the parasite, Leishmania donovani, is still able to evade the immune response of its human host. This parasite utilizes a multitude of mechanisms to disable and evade the host’s immune response.  We will focus on the mechanisms L. donovani uses to delay and evade the phagolysosomal immune response. L. donovani is able to infect phagosomes and evade the degradative properties and harsh environment created in these cells including acidic pH, increased temperatures, and increased oxidative and nitrosative stress¹. We will first start with background information about L. donovani and the life cycle of this parasite before examining how it evades the host immune system.

Leishmaniasis is a vector-borne disease caused by the unicellular, eukaryotic, obligate intracellular organism Leishmania donovani.  ‘Obligate intracellular organism’ is just a fancy way to say that it is a parasite. It means that parasites are unable to survive the conditions outside of their host because they typically rely on the cellular machinery of the host or the stable environment provided by the host. The species that cause human infection can exist in three forms.  The first is the more common cutaneous leishmaniasis form which causes skin sores².  The second main form is the visceral leishmaniasis form which infects internal organs such as the spleen, bone marrow, and liver³.  The third form is usually less common and is known as the mucosal leishmaniasis form.  This type is typically a consequence of species of the cutaneous form spreading past the skin into the mucosal membranes of the nose, throat, and mouth².

The life cycle of Leishmania donovani involves alternating between two hosts, the female phlebotomine sandfly and a human^3,4 (Fig. 1). Although there is no official starting point for the life cycle of this organism, we will start at the point at which L. donovani enters a human host. A female sandfly infected with L. donovani will bite a human to take a blood meal while simultaneously injecting L. donovani, in its promastigote stage, into the human. The promastigote cell is the mammalian-infective form of the parasite and is a thin elongated cell⁵. These cells have a flagellum which is a filament protruding from the cell that allows the parasites to move and swim around. Once these cells are injected into the human host, they swim around until they are engulfed by phagosomes in a process called phagocytosis. Phagosomes are a type of immune system cell whose function is to ingest foreign organisms and degrade them. Inside the phagosome, the promastigotes lose their flagella and ability to swim around. They transform from the promastigote cells into amastigote cells. Amastigotes are small spherical cells without a flagellum⁵. At this stage of the life cycle, amastigotes grow and divide in the phagosomes until the phagosome becomes too full and bursts, allowing the amastigote cells to infect more phagosomes². It is at this stage in the life cycle that it is determined if the human will show symptoms of infection or whether the cutaneous or visceral leishmaniasis form will develop. The form the parasite takes on usually depends on the species of L. donovani and the health of the host³. Female phleobtomine sandflies can become infected by taking a blood meal from a human infected with L. donovani. In the infected female sandflies, the phagosomes containing the amastigote cells are digested in the gut of the sandfly, releasing the amastigotes. In the midgut of the sandfly, the amastigotes transform back into the promastigote stage where they re-develop their flagella and are able to move through the midgut epithelial wall to enter the salivary glands. In the salivary glands of the sandfly, the parasites will be injected into the human host the next time the female sandfly takes a blood meal to continue the cycle².

Figure 1. Life cycle of Leishmania donovani in both the sandfly and human hosts³.    

Phagosome maturation is the process by which a phagosome containing foreign material fuses with a lysosome during phagocytosis to create a phagolysosome. It is a critical step in the killing and degradation of the ingested foreign organism⁶. Lysosomes are membrane-bound compartments containing degradative enzymes that break down the ingested material taken up by the phagosome in the mature phagolysosome.

To survive the harsh conditions in the phagosome described above, the promastigotes engulfed by the phagosome briefly prevent the maturation of the phagolysosome by expressing lipophosphoglycan (LPG) which is a sugar-phosphate molecule that is attached to the surface of the promastigotes⁷. This cell surface molecule can inhibit a variety of signaling, metabolic, and immune response molecules. It can also induce the accumulation of a structural molecule that surrounds the phagolysosome⁷. This structural component is called actin and is a single structural molecule that can link up to other actin to form large chains. The interaction between the parasite and the phagosome surface receptors, which activates the single actin molecules to assemble and surround the foreign organism, allow for the parasite to be engulfed⁷. The phagosome has a layer of actin surrounding itself that helps with engulfment of foreign organisms that slowly disappears as the phagosome goes through maturation. To be able to fully mature via fusion of the phagosome and lysosome, all of the actin needs to disappear for the fusion to occur⁷. If LPG prevents the actin from fully disappearing for a transient amount of time to allow the fusion to occur, it is able to delay the full maturation of the phagolysosome. This delay of the fusion of the phagosome and lysosome can be seen by the delay or no expression of late maturation markers normally found on the mature phagolysosome⁶. This evidence suggests that some of the surface molecules on these parasites can briefly inhibit phagosome and lysosome fusion which allows just enough time for the promastigotes to transform into the amastigotes that are capable of withstanding the degradative enzymes and acidic pH introduced by the lysosome in the mature phagolysosome⁶.

During its life cycle, Leishmania donovani is able to evade the host immune response and persist. More specifically, these parasites can survive in immune system cells known as phagolysosomes in the host, eventually killing them by bursting through the membrane to be released to infect more phagosomes.  The parasites are capable of doing this because the mammalian-infective promastigotes quickly transform into amastigotes after they are engulfed by phagosomes. The amastigotes are resistant to the degradative properties of mature phagolysosomes which allows the parasite to persist and evade the immune response.  With all of this taken together, Leishmania donovani can be an interesting pathogen for studying and understanding mechanisms of host immune system evasion.

References

1. Gupta G, et al. 2013. Mechanisms of Immune Evasion in Leishmaniasis. Advances in Applied Microbiology, 82:155-184.
2. MacMorris-Adix M. 2009. Leishmaniasis: A Review of the Disease and the Debate over the Origin and Dispersal of the Causaitive Parasite Leishmania. Macalester Reviews in Biogeography, 1(2):1-18.
3. Parasites – Leishmaniasis. Centers for Disease Control and Prevention. US Department of Health & Human Services, 2013.
4. Gossage SM, et al. 2003. Two Separate Growth Phases during the Development of Leishmania in Sand Flies: Implications for Understanding the Life Cycle. International Journal for Parasitology, 33(10):1027-1034.
5. Pulvertaft, RJ, Hoyle, GF. 1960. "Stages in the Life-cycle of Leishmania donovani". Transactions of the Royal Society of Tropical Medicine and Hygiene. 54(2):191–6.
6. Scianimanico S, et al. 1999. Impaired Recruitment of the Small GTPase Rab7 Correlates with the Inhibition of Phagosome Maturation by Leishmania donovani promastigotes. Cellular Microbiology, 1:19-32.
7. Holm A, et al. 2001. Leishmania donovani Lipophosphoglycan causes Periphagosomal Actin Accumulation: Correlation with Impaired Translocation of PKCalpha and Defective Phagosome Maturation. Cellular Microbiology, 3:439-447.

Zombie fungi and ant graveyards

by JJ

It is not unusual for parasites to control the behavior of their hosts, sometimes causing dramatic changes in their behavior: Nematode worms have been shown to cause their insect hosts to drown themselves so that their adult stages can reproduce in water ¹. Some parasitic wasp larvae can drive bumblebees to bury themselves alive to provide a better environment for the developing larvae². Similarly, the parasitic fungus Ophiocordyceps unilateralis affects its insect host’s brain, effectively turning the insect into a ‘zombie’ whose only purpose is to serve as food for the fungus and a vehicle to carry it to more favorable environments.

O. unilateralis was first described by English scientist Tom Petch in 1931 as a top-shaped fungus found on dead aphids³(A common sap-sucking insect pest). He also noted how all dead aphids infected by the fungus were found ‘glued’ to leaves by fungal protrusions³. Since then, O. unilateralis has been shown to infect many species of insects, but especially ants in tropical forests around the world. There are even distinct species found in China, South America, and Africa^4,5. These fungi are thought to help keep insect populations in check. They have also been used in ancient Chinese medicine for hundreds of years, although they have only recently been investigated for the production of therapeutic compounds⁶.

When an ant is infected by O. unilateralis while foraging, it will eventually abandon all ant-like behavior, its mind seemingly under the control of the fungus: hence the term ‘zombie fungus’. When the ant walks over fungal spores, the spores cling to the exoskeleton (a hard shell-like outer layer) of the ant. Within 3 days, the spores germinate and reproduce within the ant⁷. In the meantime, the ant’s behavior starts to change. It stops foraging and following scent trails altogether and starts to climb to locations close to the forest floor, such as leaves. Once it reaches a leaf, the ant uses its mandibles (mouthparts) to clamp on to the leaf and stays put until it eventually dies of starvation, the fungus having used its body to reproduce. It is not clear how the ant’s colleagues feel about this.

Once the ant dies, the fungus produces a long stalk emerging from the back of the ant’s head (Fig. 1). This stalk bears a structure filled with new fungal spores. Eventually, these spores drop to the forest floor, where they can infect new insects and continue the cycle. The spread of spores in this manner means infected insects are often found clustered around the same area, forming ‘graveyards’⁷. Researchers have undertaken the grim task of studying different conditions surrounding these graveyards over time and have found that they are more likely to be present where humidity is high and foliage is dense- conditions that would give the fungus a better chance to reproduce. The number of graveyards has also been found to increase during the monsoons, and decrease in the summers⁷. More importantly, other insects have been shown to actively avoid these graveyards, and ant trails very rarely cut across graveyards⁷. Ant colonies prefer foraging up the treetops and minimize time spent on the forest floor⁷. They are also known to groom each other, which is thought to help remove spores and keep the colony immune to infection⁸. These behaviors have probably evolved as defense mechanisms against fungal infection, and suggest that the fungus has co-evolved with insects, with insects being evolutionarily selected for avoiding the fungus, and the fungus evolving to more efficiently disperse its spores⁵.

There are other aspects of O. unilateralis infection that are yet to be fully understood. For example, the interactions between the parasite and the host at the molecular level are not yet clear⁹. These are important because they would give us a clearer picture of how the fungus proceeds from the spore to the stalk-forming stage during infection. Next, the effect that the fungus has on the insect nervous system also needs to be described, aside from just behavioral changes. The full range of behavioral changes induced in ants is also not clear: some researchers suspect that the fungus induces aggressive behavior in ants, which could help the spores spread to other workers in the ant colony⁹. Using large indoor ant colonies for observational studies (Fig. 2) would allow researchers to manipulate ants and isolate each of these behavioral and physiological changes, helping to paint a more complete picture of the zombie fungus.

References                                                                                          

1.         Sato, T., Watanabe, K., Tamotsu, S., Ichikawa, A. & Schmidt-Rhaesa, A. Diversity of nematomorph and cohabiting nematode parasites in riparian ecosystems around the Kii Peninsula, Japan. Can. J. Zool. 90, 829–838 (2012).

2.         Müller, C. B. Parasitoid induced digging behaviour in bumblebee workers. Anim. Behav. 48, 961–966 (1994).

3.         Petch, T. Notes on entomogenous fungi. Trans. Br. Mycol. Soc. 16, 55–75 (1931).

4.         Evans, H. C., Elliot, S. L. & Hughes, D. P. Hidden Diversity Behind the Zombie-Ant Fungus Ophiocordyceps unilateralis : Four New Species Described from Carpenter Ants in Minas Gerais, Brazil. PLOS ONE 6, e17024 (2011).

5.         Kobmoo, N., Mongkolsamrit, S., Tasanathai, K., Thanakitpipattana, D. & Luangsa-Ard, J. J. Molecular phylogenies reveal host-specific divergence of Ophiocordyceps unilateralis sensu lato following its host ants. Mol. Ecol. 21, 3022–3031 (2012).

6.         Isaka, M., Kittakoop, P., Kirtikara, K., Hywel-Jones, N. L. & Thebtaranonth, Y. Bioactive substances from insect pathogenic fungi. Acc. Chem. Res. 38, 813–823 (2005).

7.         Pontoppidan, M.-B., Himaman, W., Hywel-Jones, N. L., Boomsma, J. J. & Hughes, D. P. Graveyards on the Move: The Spatio-Temporal Distribution of Dead Ophiocordyceps-Infected Ants. PLOS ONE 4, e4835 (2009).

8.         Schmid-Hempel, P. Parasites in Social Insects. (Princeton University Press, 1998).

9.         Bekker, C. de, Merrow, M. & Hughes, D. P. From Behavior to Mechanisms: An Integrative Approach to the Manipulation by a Parasitic Fungus (Ophiocordyceps unilateralis s.l.) of Its Host Ants (Camponotus spp.). Integr. Comp. Biol. icu063 (2014). doi:10.1093/icb/icu063