Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi

Abstract

Background: Brugia malayi and B. pahangi are two closely related nematodes that cause filariasis in humans and animals. However, B. pahangi microfilariae are able to develop in and be transmitted by the mosquito, Armigeres subalbatus, whereas most B. malayi are rapidly melanized and destroyed within the mosquito hemocoel. A cross-species microarray analysis employing the B. malayi V2 array was carried out to determine the transcriptional differences between B. malayi and B. pahangi microfilariae with similar age distribution.

Results: Following microarray data analysis, a list of preferentially expressed genes in both microfilariae species was generated with a false discovery rate estimate of 5% and a signal intensity ratio of 2 or higher in either species. A total of 308 probes were preferentially expressed in both species with 149 probes, representing 123 genes, in B. pahangi microfilariae and 159 probes, representing 107 genes, in B. malayi microfilariae. In B. pahangi, there were 76 (62%) up-regulated transcripts that coded for known proteins that mapped into the KEGG pathway compared to 61 (57%) transcripts in B. malayi microfilariae. The remaining 47 (38%) transcripts in B. pahangi and 46 (43%) transcripts in B. malayi microfilariae were comprised almost entirely of hypothetical genes of unknown function. Twenty-seven of the transcripts in B. pahangi microfilariae coded for proteins that associate with the secretory pathway compared to thirty-nine in B. malayi microfilariae. The data obtained from real-time PCR analysis of ten genes selected from the microarray list of preferentially expressed genes showed good concordance with the microarray data, indicating that the microarray data were reproducible.

Conclusion: In this study, we identified gene transcripts that were preferentially expressed in the microfilariae of B. pahangi and B. malayi, some of which coded for known immunomodulatory proteins. These comparative transcriptome data will be of interest to researchers keen on understanding the inherent differences, at the molecular level, between B. malayi and B. pahangi microfilariae especially because these microfilariae are capable of surviving in the same vertebrate host but elicit different immune response outcomes in the mosquito, Ar. subalbatus.

Figure 1

Sequence alignment of five probes against the PCR products of B. pahangi and B. malayi. Primers designed to flank the probe sequence of five probes on the BmV2 chip that had three to ten times higher signal intensities in B. malayi microfilariae than in B. pahangi were used for PCRs with B. pahangi and B. malayi genomic DNA as templates and the PCR product was sequenced. The asterisks indicate identical nucleotides in the aligned sequences. For Bm1_34045, the consensus dinucleotides at either ends of the intron, GT at the 5' end and AG at the 3' end, are shown in bold for both the B. pahangi and B. malayi genes.

Figure 2

KEGG pathway classification of preferentially expressed genes in B. pahangi and B. malayi microfilariae. The genes that were classified as up-regulated in either species were manually mapped into the KEGG pathway groups. Cellular; Cellular Processes, Environmental; Environmental Information Processing, Genetic; Genetic Information Processing.

Figure 3

Comparison of real-time PCR and microarray data of ten randomly selected genes. Real-time PCR analysis was performed on cDNA derived from five biological samples of B. pahangi and B. malayi microfilariae with similar age distribution. (A) The ratio of Ct values obtained following real-time PCR analysis using primers flanking three probes shown to have similar signal intensities in the microarray data. Probe bm.02018, which represented Pub locus Bm1_34045, was assigned as the endogenous control gene. (B) Comparison of log-transformed microarray data (three biological samples) and real-time PCR data (five biological samples) of the ten representative genes showing good agreement of expression values obtained from both methodologies. Biological samples used for real-time PCR analysis were independent of those used for microarray analysis.