The transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design

Abstract

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.

De Novo Transcriptome Profiling of Naegleria fowleri Trophozoites and Cysts via RNA Sequencing

“…[ 39 , 40 ]. Recently, studies of the transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design and treatment with Hesperidin conjugated with silver nanoparticles using RNA-Seq in N. fowleri infection were reported [ 41 , 42 ]. In a previous study, Nf-actin was located in the cytoplasm, pseudopodia, and amoebastome in N. fowleri trophozoites [ 43 ].…”

Glucose metabolism in the pathogenic free‐living amoebae: Tempting targets for treatment development

“… a Proteins identified with lowest E value score after query using human orthologs against AmoebaDB (https://amoebadb.org/amoeba/app/) or against the Balamuthia mandrillaris transcriptome (Phan et al., 2021). …”

“…This enzyme is responsible for catalyzing the conversion of DHAP to glyceraldehyde 3-phosphate (G3P Proteins identified with lowest E value score after query using human orthologs against AmoebaDB (https:// amoeb adb. org/ amoeba/ app/ ) or against the Balamuthia mandrillaris transcriptome (Phan et al, 2021).…”

Distribution and Current State of Molecular Genetic Characterization in Pathogenic Free-Living Amoebae

“…Nevertheless, metagenomic analysis has been used to diagnose this pathogenic protozoan in samples from sick patients; but since it is a disease with a high mortality rate, unfortunately, most of them did not survive [ 16 , 115 , 128 , 168 ]. In addition, the development of new compounds against B. mandrillaris has been limited by the scarcity of genomic information, so a recent study performed a transcriptome analysis with the identification of 17 target genes, and 15 were validated by direct sequencing; these genes would be helpful to develop new approaches for the treatment of B. mandrillaris infections [ 169 ]. For this pathogen, in particular, it is necessary to expand the information on the omics technologies because it can remain in the host for years [ 131 ], in addition to presenting characteristics such as difficulty in its identification employing conventional techniques (molecular, microbiological, or histopathological) and the disease presenting different times of expression.…”