Reverse Epidemiology: An Experimental Framework to Drive Leishmania Biomarker Discovery in situ by Functional Genetic Screening Using Relevant Animal Models

Abstract

Leishmania biomarker discovery remains an important challenge that needs to be revisited in light of our increasing knowledge on parasite-specific biology, notably its genome instability. In the absence of classical transcriptional regulation in these early-branching eukaryotes, fluctuations in transcript abundance can be generated by gene and chromosome amplifications, which have been linked to parasite phenotypic variability with respect to virulence, tissue tropism, and drug resistance. Conducting in vitro evolutionary experiments to study mechanisms of Leishmania environmental adaptation, we recently validated the link between parasite genetic amplification and fitness gain, thus defining gene and chromosome copy number variations (CNVs) as important Leishmania biomarkers. These experiments also demonstrated that long-term Leishmania culture adaptation can strongly interfere with epidemiologically relevant, genetic signals, which challenges current protocols for biomarker discovery, all of which rely on in vitro expansion of clinical isolates. Here we propose an experimental framework independent of long-term culture termed “reverse” epidemiology, which applies established protocols for functional genetic screening of cosmid-transfected parasites in animal models for the identification of clinically relevant genetic loci that then inform targeted field studies for their validation as Leishmania biomarkers.

The critical role of mode of action studies in kinetoplastid drug discovery

“…By combining chemical mutagenesis with nextgeneration sequencing, known as Mut-Seq, can lead to the rapid identification of mutations associated with drug resistance. However, it should be born in mind that not all resistance mechanisms are likely to arise readily in clinical situations due to drug pharmacodynamic behaviour in vivo, fitness costs and transmission potential associated with a resistance phenotype (Piel et al, 2018;Reis-Cunha et al, 2018;Van Bockstal et al, 2020).…”

Aportes Y Dificultades De La Metagenómica De Suelos Y Su Impacto en La Agricultura.

“…Sin importar el ambiente o ecosistema a estudiar existen tres pasos importantes en la realización de un estudio metagenómico; el primero es la colecta de la muestra de interés, el segundo es la extracción del ADN total de la muestra y el tercero son las tres diferentes opciones que existen para analizar la muestra. A) El ADN metagenómico puede ser sometido a un proceso de digestión y clonación en vectores de expresión como los denominados cromosomas artificiales de bacterias (BAC), cromosomas artificiales de levadura (YACs), Cósmidos, o Fósmidos, que nos permiten construir librerías metagenómicas recuperando insertos de ADN mayores a 40 Kb (Jung y Kim, 2018;Piel et al, 2018;Tocchetti et al, 2018;Buckley y Ettensohn, 2019). B) amplificación por PCR de los genes que codifican para los ARN´s ribosomales (ARN´s) 16S o 18S, lo cual permite conocer la diversidad de microorganismos en la muestra (bacterias, arqueas o eucariontes) para realizar un análisis filogenético.…”