The Elucidation of Drug Resistance Mechanisms for

by JH

Figure 1: An infection with the bone marrow

 of L. donovai as extracellular amastigotes (A). 

As you can see the polynuclear phagocyte in

the top right is being lysed to release the

amastigotes for further infection. (AN) represents

the amastigote nucleus. 

Visceral leishmanisis is a protozoan systemic infection which is estimated to infect 500,000 people annually, and kill 50,000 if left untreated. The infection is caused by over twenty different species, but the most virulent and deadly is caused by the obligatory intracellular protozoan, Leishmania donovai.¹ This nasty parasite is transmitted through hematophagous sandflies, and like many other insect vectored parasites is especially prevalent in poverty stricken areas such as East Africa, Nepal and India. L. donovai is injected into a human host from the sandfly’s salivatory gland as a promastigote. The parasite invades host macrophages and neutrophils to replicate as amastigotes (Figure 1). From there, the parasite overruns and infects additional macrophages to eventually become a systemic infection as they reach the host’s bone marrow, liver, and spleen. Because of the decimation and destruction of critical host immune cells and pleuripotient stem cells, a secondary infection is the usual cause of death¹.

The most current treatment option for patients with leishmaniasis other than costly chemotherapy treatments is pentavalent antimonials [Sb(V)]. Although the drug is often efficient at treating the infection and has been in clinical use for several decades, high rates of antimonial resistance have been reported.^1-3 The resistance to Sb(V) poses an interesting question, by which mechanism does L. donovani acquire resistance to Sb(V), and is this mechanism/mechanisms conserved throughout the species?  One model from a Belgian research group is that the parasite changes its thiol metabolism which would inhibit the activation of Sb(V). The parasite also employs a blocking mechanism in which decreased expression of a specific transmembrane protein (AQ1) doesn’t allow for the uptake of essential anti-parasitic drugs. A second theory of resistance by Neeloo Singh from the Central Drug Research Institute in Lucknow India is that the parasite evades the cytotoxic effects of Sb(V) therapy though the enhanced efflux of drugs due to an overexpression of transmembrane proteins belonging to the superfamily of ABC transporters. The elucidation of resistance mechanisms is key to the design of new efficient drugs, and for the creation of a potential vaccine against the parasite.  

The first aforementioned mechanistic model of Sb(V) resistance that was proposed by a Belgian group is addressed in Gene Expression Analysis of the Mechanism of Natural Sb(V) Resistance in Leishmania donovani Isolates from Nepal². What they were trying to do was elucidate the mechanism of natural Sb(V) resistance of L.donovani strains isolated from infected patients. To do this, they analyzed expression profiles of all previously known genes putatively involved in Sb(V) metabolism.  Through real-time quantitative PCR, differential gene expression profiles were created in natural Sb(V)-resistant and sensitive L.donovani  strains. These strains demonstrated significantly reduced expression levels for thee different genes: 1) gcs, coding for the enzyme that catalyzes the rate-limiting step in the synthesis of glutathione; 2) odc coding for the enzyme that catalyzes the rate-limiting step of the synthesis of the spermidine moiety of trypanothione; and 3) aqp1, a gene that codes for the protein responsible for the uptake of Sb(V). A lower expression of thiol biosynthetic enzymes (GCS and ODC) leads to a decreased rate of thiol biosynthesis and is believed to inhibit the activation of Sb(V) inside of  amastigotes². This is an essential mechanism because thiols have been previously shown to sensitize amastigotes to Sb(V) as they promote the activation and interaction of the drug^1-2.  Additionally, the lowering of aqp1 expression causes a decrease in the essential AQP1 membrane transport protein that has been previously shown to be one of the main receptors and channels for Sb(V) entry into the cell.²

Although this research is interesting and elucidating, there are discrepancies within the data set that need to be addressed. First and foremost, they note that in intracellular amastigotes, all three genes had lower level of expression in Sb(V)-resistant strains than in sensitive strains. But, in promastigotes, the differences between resistant and sensitive strain gene expression were much less pronounced for GCS and AQP1 and completely ablated for ODC.  This may imply that the expression of resistance genes are due to different regulatory developmental controls within the amastigotes and promastigotes that help differentiate resistance gene expression at different times of the infection cycle.  

Another mechanism proposed in Drug resistance mechanisms in clinical isolates of Leishmania donovani by Neelo Singh is the enhanced efflux of drugs through over expressed membrane proteins belonging to the superfamily of ABC transporters³. Through microarray analysis of cDNAs from both promastigote and amastigote stages of the parasite’s life, Singh was able to show that when compared to clinical Sb(V) sensitive strains of L. donovani, the Sb(V) resistant strains had highly expressed levels of  ABC transporter genes. The ABC transporter proteins have been extensively studied in the past for their role in drug resistance. Due to their efflux capabilities, they work within the plasma membrane to pump out and remove unwanted compounds (such as drug compounds) from the cellular cytoplasm.^{1, 3} In addition, a specific ABC transporter pump MRP, was detected to have increased activity when assayed through flow cytometry. What is interesting and is noted in this paper is that trypanothione binds to Sb(V) and a specific set of ABC transporters has been previously shown to exclusively bind to trypanothione and efflux it from the cell.³ This notion of trypanothione being a major player in resistance is also supported in the paper from the Belgium group in that trypanothione binds to Sb(V) to activate the drug. This leads to the formation of an interesting idea that the parasite has developed multiple mechanisms for either the reduction of trypanothione production and/or for the increase of specific ABC transporters to efflux both trypanothione (a molecule unique towards Kinetoplastida, which is the class that L. Donovani resides within) and Sb(V) drug out of the cell.³

One limitation that I found within this data is that the three isolates used in the study all were found to have varying levels of resistance when tested for antimonials drug sensitivity in vitro. By testing isolates with different drug sensitivities we cannot correctly identify  conserved gene expression levels through microarray test and flow cytometry due to higher gene expression by more resistant isolates and vice versa.

In conclusion, it appears that the parasite L.donovai employs several different mechanisms to become resistant and survive against the pressures of antimonial drugs. These two papers propose that 1) an increase in ABC transporter gene expression would result in the amplification of Sb(V) efflux; 2) decreased apq1 gene expression would inhibit the cell from taking up Sb(V); and 3) the parasite mounts a two sided attack on thiols to either expel them from the cell (though increased thiol-specific ABC transporter gene expression), or by lowering the expression of the  odc gene that participates in thiol synthesis and thus inhibits the synthesis of thiol compounds such as trypanothione. The overall phenomenon of antimonial resistance appears to be multifactorial and we would need to apply multiple therapeutics that target several metabolic pathways within the parasite. For this to occur we need to further research L. donovai cell metabolism and mechanical pathways to gain a better understanding of their ability to resist modern day drugs. If preformed, the scientific community as a whole will be better adept to use and create drug cocktails that will be able to inhibit elusive parasitic resistant pathways, and hopefully eradicate the effects of Leishmania from the earth.

Selected References

1. Maltezou, Helena C. "Drug Resistance in Visceral Leishmaniasis." National Center for Biotechnology Information. U.S. National Library of Medicine, 01 Nov. 2009. Web. 15 Nov. 2012. 
2. Decuypere, S. et al. Gene expression analysis of the mechanism of natural Sb(V) resistance in Leishmania donovani isolates from Nepal. Antimicrob. Agents Chemother. 49, 4616–4621 (2005).
3. Singh, N. Drug resistance mechanisms in clinical isolates of Leishmania donovani. Indian J. Med. Res. 123, 411–422 (2006).
4. Bueno, M. Herrares, J. Visceral Leishmaniasis. New England Journal of Medicine 336: 13, 965-966, (1996)