Gene expression profiling ofLeishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation

Abstract

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to ' freeze' gene expression levels instantly ; this is particularly a delicate task for intracellular amastigotes and for specific ' sensory ' genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.

Comparison of gene expression patterns amongLeishmania braziliensisclinical isolates showing a differentin vitrosusceptibility to pentavalent antimony

“…Indeed, this latter model already implied overexpressed genes in Sb V -resistant L. donovani isolates (Decuypere et al 2005;Mukherjee et al 2007). However, this life stage is extremely sensitive to minimal disturbance of the parasite's environment during harvesting, hence extreme care is needed to 'freeze' gene expression levels instantly (Decuypere et al 2008). In that sense, axenic amastigotes might represent an interesting biological compromise between promastigotes and intracellular amastigotes, but axenization is not straightforward for some species and for clinical isolates.…”

“…These markers are not yet available, due to the limited knowledge of the molecular and biochemical mechanisms underlying resistance to Sb V , especially in clinical isolates. Even if intracellular amastigotes may be considered as the clinically relevant stage, the current protocols to study their gene expression require extreme care to ensure reproducibility of the results (Decuypere et al 2008) and are not adapted to the study of a large number of isolates. In contrast, promastigotes can be easily cultivated and harvested with minimal risk of affecting their biological state under well-controlled in vitro conditions (Decuypere et al 2008).…”

“…Even if intracellular amastigotes may be considered as the clinically relevant stage, the current protocols to study their gene expression require extreme care to ensure reproducibility of the results (Decuypere et al 2008) and are not adapted to the study of a large number of isolates. In contrast, promastigotes can be easily cultivated and harvested with minimal risk of affecting their biological state under well-controlled in vitro conditions (Decuypere et al 2008). Furthermore, molecular changes during promastigote differentiation serve as pre-adaptations for parasite transmission and initial infection stages in the vertebrate host (Sacks, 1989;Saxena et al 2003).…”

“…Hence, a time-course analysis of gene expression changes throughout the promastigote growth cycle may show differences in molecular adaptations of parasites to intracellular life. Our previous study on L. donovani clinical isolates indicated that promastigote expression-curves could indeed reveal molecular features distinguishing isolates with different in vitro Sb V susceptibility phenotypes (Decuypere et al 2008). Moreover, considering that the in vitro growth rates could vary among different isolates, expression-curves are advisable to guarantee a reliable molecular comparison of identical developmental stages among various isolates under study.…”

Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance

“…These populations constitute an ideal genomic model in which genetic variation between samples should be reduced: Indeed, these parasites are relatively homogeneous, likely as a result of a demographic bottleneck in the 1960s, after DDT spraying campaigns (WHO Expert Committee on the Control of the Leishmaniases 2010). In addition, these isolates were the subjects of several recent studies, providing a unique clinical, biological, and epidemiological background (Decuypere et al 2005(Decuypere et al , 2008Laurent et al 2007;Alam et al 2009;Rijal et al 2010;Vanaerschot et al 2010). Importantly, these parasites are highly variable in terms of susceptibility to antimonial drugs such as sodium stibogluconate (SSG; Rijal et al 2007;Samant et al 2007;Kumar et al 2009).…”