Mechanisms of Cholesterol Sabotage and Dicer Assassination in Leishmania donovani

by TB

When many people hear the terms assassination and sabotage the first thing that comes to mind is almost certainly a James Bond movie or some other stereotypical spy flick. In this movie, the dashing charismatic lead infiltrates an enemy stronghold to hinder the plans of some nefarious villain who plans to dispose of the hero in unforetold ways. Well, it has often been said that the truth is stranger than fiction and in the case of Leishmania donvani, it is certainly the case.

Figure 1. Severe splenomegaly or

enlargement of the spleen due

to visceral leishmaniasis.   

In order to understand the acts of assassination and sabotage that this organism performs it helps to have a little background information. Leishmania donovani is a protozoan parasite that is the causative agent of visceral leishmaniasis in humans. Visceral leishmaniasis (VL) is a systemic infection meaning that it affects many tissues and organs within the body. This infection usually occurs after a 2-6 month incubation period at which time symptoms correlating to systemic infection and invasion of the blood occur. These symptoms may include fever, weakness, loss of appetite, weight loss, as well as the enlargement of the lymph nodes, spleen, and liver. At later stages of infection, severe enlargement of the spleen and liver may possibly be observed on a macroscopic level (Fig. 1). The symptoms of LV can persist for weeks to months and may lead to massive bleeding, severe anemia, and death if left untreated (1).
Every year approximately 500,000 new cases of VL are recorded and approximately 50,000-70,000 deaths occur due to this disease. It has been reported that the only parasitic disease with a higher death toll is malaria. Most of these cases (>90%) have been recorded in Bangladesh, India, Nepal, Sudan, Ethiopia, and Brazil (1, 2).  It is disheartening to relay that areas that are endemic to VL are often rural and associated with poverty. Due to this disease families often spend large amounts of money on treatment and often lose income due to the infected individual’s inability to work (1, 2) (Fig. 2).

Figure 2. Reported new cases of VL according to the WHO as of 2013. A majority of cases occur in Bangladesh, India, Nepal, Sudan, Ethiopia, and Brazil.   

At this point, it is pretty obvious that Leishmania donovani isn’t the “good guy” in this scenario. In fact, this parasite is more like an undercover agent pulling strings from behind the scenes to benefit itself at the expense of its’ victims. The question is: how did Leishmania donovani implant itself into humans in order to benefit itself and cause disease?

Leishmania species are typically transferred between hosts in the gut of a female phlebotomine sand fly. Within this sand fly, Leishmania are typically in a promastigote form. The promastigote form is characterized by having long cells that are able to move using a flagellum within the fly (1–3). Within the sand fly Leishmania species replicate and prepare for the invasion of a new host (2). When the female sand fly finds an unfortunate individual deemed worthy of her next meal the promastigotes are transferred into a new human host. Once inside a host, the promastigotes migrate to the liver. The presence of the parasite activates an immune response in the human host. One of the first human immune cells that reacts to this new infection is the macrophage. The macrophage identifies the promastigote as an invader and tries to surround and destroy it using a process called phagocytosis. During this process, promastigotes use a variety of strategies to avoid being killed by the macrophages. Some of these strategies include preventing the release of deadly antimicrobial agents and the inhibition of other macrophage functions that are required for an immune response to the parasite (3). These activities allow for the survival of the parasite within the macrophage. Differentiation of the promastigote form into the amastigote form occurs next. The amastigote form is characterized by the loss of the flagella and round cells (1–3). The amastigotes then replicate within the disabled macrophage (1). The multiplication and spread of amastigotes throughout the body is what leads to typical LV symptoms and systemic infection. After spreading throughout the body another female phlebotomine sand fly can then become infected and have the amastigotes differentiate into the promastigote form to repeat the cycle (Fig. 3).

Figure 3. The life cycle of Leishmania species. Transmission

occurs from female sandflies while in the promastigote form.

Change into the amastigote form occurs in a human host.

Multiplication of amastigotes leads to characteristic VL symptoms.

Right now you are probably asking how any of this information has anything to do with assassination or sabotage and why the title was so misleading. Well, if you remember the ability of Leishmania to inhibit the function of macrophages and survive within them you have your answer. One of the mechanisms for the survival of Leishmania donovani within macrophages is the metaphorical assassination of the host protein named Dicer. This assassination leads to the sabotage of cholesterol metabolism in human host cells in the liver to make infection easier. To understand how the assassination of Dicer leads to the sabotage of cholesterol metabolism it is important to understand how Dicer plays a part in the regulation of cholesterol metabolism.

Dicer is a protein that normally plays a role in the creation of micro RNAs. A micro RNA (miR) is an RNA that is created during the transcription of a DNA sequence to an RNA sequence. Unlike many RNAs transcribed, miRNAs do not code for a protein sequence and are often referred to as noncoding RNAs. Although these noncoding RNAs do not play a role in the formation of protein they have been observed to be involved in post-transcriptional regulation. This means that they play a role in the regulation of gene expression and help to control the amounts of proteins that are made from a specific gene. The use of miRNAs in post-transcriptional regulation starts with the creation of a DNA sequence in the nucleus that codes for a noncoding miRNA. These newly formed miRNAs are called pri-miRNAs and form the hairpin loop structures shown in Figure 4 (4). The newly formed pri-miRNAs interact with a protein complex made up of two proteins called Drosha and Pasha (aka DGCR8). The protein complex containing Drosha and Pasha is used to cleave loose hanging pri-miRNA ends and process them so they are better suited for their jobs. After the interaction with the Drosha and Pasha protein complex, the miRNAs are referred to as pre-miRNAs. The newly formed pre-miRNAs still have a hairpin loop structure and are then transported out of the nucleus where they encounter the Dicer protein. Dicer’s role is to cleave the loop portion of the hairpin structure and to further fine tune the pre-miRNAs for their regulatory jobs. After the interaction with Dicer, the pre-miRNAs are now considered full-fledged miRNAs. These miRNAs are loaded onto RISC protein complexes. These RISC complexes escort the miRNAs around various areas in the cell to perform regulation activities. Now that we understand how Dicer normally functions to help in the creation of miRNAs the assassination of Dicer and sabotage of cholesterol metabolism can be examined.

Figure 4. Mechanism of Dicer cleavage as described by Descoteaux and colleagues (4). The GP-63 protease cleaves Dicer to prevent the maturation of miR-122 pre-RNA. This causes a decrease in the levels of cholesterol within the human host. This allows for Leishmania to replicate and spread through the body easier.

Normally, the regulation of cholesterol metabolism in the liver is partially controlled by the post-transcriptional control using a miRNA called miR-122 (5).  During infection with Leishmania donovani, a protein called a GP-63 protease has been shown to be used to cleave Dicer (6). Since Dicer is no longer functional miR-122 copies can no longer mature or function to regulate cholesterol metabolism (6). The loss of functional copies of miR-122 leads to lower levels of cholesterol in the serum of human host. This has been shown to be beneficial for the invasion of Leishmania donovani and allows for further infection of liver cells in a human host (7). The next question to ask is how the GP-63 protease moves from a Leishmania donovani parasite within a macrophage to a human liver cell to cleave Dicer. Unfortunately, the process by which this happens is unknown and the undercover agent still remains behind the scenes of the crime pulling the strings for its’ own benefit.

Before you leave thinking that all hope is lost and that the undercover agent will forever remain in the shadows of the macrophage pulling the strings to our liver cells for eternity let us have a debrief. The undercover operative Leishmania donovani is a protozoan parasite. It is also the main causative factor of the disease visceral leishmaniasis which is primarily observed in Bangladesh, India, Nepal, Sudan, Ethiopia, and Brazil. The only reason this disease is able to manifest is due to the stealth and techniques employed by Leishmania donovani to evade the immune system and facilitate infection. The most notable of these techniques is the assassination of Dicer using a GP-63 protease. This assassination ultimately leads to the sabotage of cholesterol metabolism within the unsuspecting victim and allows for Leishmania donovani to replicate, spread, and cause disease easier. Although the process by which this undercover agent performs this assassination and sabotage is still shrouded in mystery there is no need to fear! There is still hope that the scientific community will finally discover how to purge this menace from the depths of unsuspecting macrophages and free us from this disease. So carry on fellow microbiology enthusiast and fear not the undercover agent that invades, assassinates, and sabotages for the future only holds more scientific discovery and with it the information required to expunge the undercover agent named Leishmania donovani.

Citations

1.        Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, Alvar J, Boelaert M. 2007. Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol 5:S7–S16.

2.        Murray HW, Berman JD, Davies CR, Saravia NG. 2005. Advances in leishmaniasis. Lancet 366:1561–1577.

3.        Olivier M, Gregory DJ, Forget G. 2005. Subversion mechanisms by which Leishmania parasites can escape the host immune response: A signaling point of view. Clin Microbiol Rev. American Society for Microbiology.

4.        Descoteaux A, Moradin N, Arango Duque G. 2013. Leishmania dices away cholesterol for survival. Cell Host Microbe 13:245–247.

5.        Wen J, Friedman JR. 2012. miR-122 regulates hepatic lipid metabolism and tumor suppression. J Clin Invest 31:2773–2776.

6.        Ghosh J, Bose M, Roy S, Bhattacharyya SN. 2013. Leishmania donovani targets dicer1 to downregulate miR-122, lower serum cholesterol, and facilitate murine liver infection. Cell Host Microbe 13:277–288.

7.        Ghosh J, Das S, Guha R, Ghosh D, Naskar K, Das A, Roy S. 2012. Hyperlipidemia offers protection against Leishmania donovani infection: role of membrane cholesterol. J Lipid Res 53:2560–2572.