The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi

Abstract

Background: Filarial nematodes cause serious and debilitating infections in human populations of tropical countries, contributing to an entrenched cycle of poverty. Only one human filarial parasite, Brugia malayi, can be maintained in rodents in the laboratory setting. It has been a widely used model organism in experiments that employ culture systems, the impact of which on the worms is unknown.

Methodology/principal findings: Using Illumina RNA sequencing, we characterized changes in gene expression upon in vitro maintenance of adult B. malayi female worms at four time points: immediately upon removal from the host, immediately after receipt following shipment, and after 48 h and 5 days in liquid culture media. The dramatic environmental change and the 24 h time lapse between removal from the host and establishment in culture caused a globally dysregulated gene expression profile. We found a maximum of 562 differentially expressed genes based on pairwise comparison between time points. After an initial shock upon removal from the host and shipping, a few stress fingerprints remained after 48 h in culture and until the experiment was stopped. This was best illustrated by a strong and persistent up-regulation of several genes encoding cuticle collagens, as well as serpins.

Conclusions/significance: These findings suggest that B. malayi can be maintained in culture as a valid system for pharmacological and biological studies, at least for several days after removal from the host and adaptation to the new environment. However, genes encoding several stress indicators remained dysregulated until the experiment was stopped.

Fig 1. Study design.

At each time point, two groups of 8 worms from each jird were washed, flash-frozen and used for RNA extraction.

Fig 2. Venn diagram showing the number of overlapping genes across pairwise comparisons of each time point compared to baseline.

Venn diagram was created using Venny 2.0.

Fig 3. Differentially expressed genes in each time point comparison, with the highest fold-changes.

A log₂ fold-change of +3.5 and -3.5 was applied, respectively. GO terms were retrieved from Nematode.net. Green indicates down-regulation and red, up-regulation.

Fig 4. Timewise comparison of gene expression.

A. Heat map showing pairwise comparison of genes from GO enrichment analysis. B. Heat map showing pairwise comparison of genes found in enrichment pathway analysis. Genes were normalized using the trimmed mean of M-values normalization method in edgeR and clustered according to Euclidean distance metrics. Green indicates relative down-regulation and red relative up-regulation. Values on the X-axis represent the sample identifications at each time point. Each sample identification is coded by the jird number from which it originated, the time point at which it was analyzed, and the sample replicate letter (a or b).

Fig 5. Correlation between RNAseq and qPCR data from 5 genes at different time points.

Fold-change values of the selected genes are displayed in S1 Table. The correlation coefficient between RNAseq (x-axis) and qPCR (y-axis) data (log₂ fold-change) analyzed by the Pearson test was 0.9961 with a statistical significance p<0.01.