Unknown,Transcriptomics,Genomics,Proteomics

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Dual RNA-seq of parasite and host reveals gene expression dynamics during filarial worm-mosquito interactions

ABSTRACT: Parasite biology, by its very nature, cannot be understood without integrating it with that of the host, nor can the host response be adequately explained without considering the activity of the parasite. However, due to experimental limitations, molecular studies of parasite-host systems have been predominantly one-sided investigations focusing on either of the partners. Here we conduct a joint dual RNA-seq time course analysis of filarial parasite and host mosquito to better understand the parasite processes underlying development in, and interaction with, the host tissue from the establishment of infection to the emergence of infective-stage larva. Using the Brugia malayi-Aedes aegypti system, we report the parasite gene transcription dynamics, which exhibit a highly ordered developmental program consisting of a series of cyclical and state-transitioning temporal patterns. And, we contextualize these parasite data in relation to the concurrent dynamics of the host transcriptome. Comparative analyses using uninfected tissues and different host strains reveal the influence of parasite development on the host gene transcription as well as the influence of host environment on the parasite gene transcription. Furthermore, we critically evaluate the life-cycle transcriptome of B. malayi by comparing developmental stages in the mosquito relative to those in the mammalian host, providing insight into gene expression changes underpinning the mosquito-borne parasitic lifestyle of this heteroxenous parasite. Time-course mRNA profiles of filarial parasite Brugia malayi and host mosqutio Aedes aegypti were generated by deep sequencing using Illumina GAIIx.

ORGANISM(S): Aedes aegypti

SUBMITTER: Bruce Christensen

PROVIDER: E-GEOD-53664 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mosquito Anopheles gambiae uses its innate immune system to control bacterial and Plasmodium infection of its midgut tissue. The activation of potent IMD pathway-mediated anti-Plasmodium falciparum defenses is dependent on the presence of the midgut microbiota, which activate this defense system upon parasite infection through a peptidoglycan recognition protein, PGRPLC. We employed transcriptomic and reverse genetic analyses to compare the P. falciparum infection-responsive transcriptomes of septic and aseptic mosquitoes and to determine whether bacteria-independent anti-Plasmodium defenses exist. To examine the impact of P. falciparum infection on the mosquito midgut and carcass transcriptomes in the presence or absence of midgut bacteria, we used A. gambiae whole genome microarrays to compare the mRNA abundance of P. falciparum-infected and -naÃ¯ve mosquitoes of antibiotic- and non-antibiotic treated cohorts. P. falciparum infection induced changes in the abundance of as many as 2,183 and 2,429 transcripts in whole mosquitoes belonging to a variety of functional groups in aseptic and septic mosquitoes. Ultimately, we were interested in identifying the genes involved in bacteria-independent anti-Plasmodium responses, and therefore we focused on transcripts displaying increased abundance in the parasite-infected aseptic midguts, placing a particular emphasis on those with predicted immune functions. Because of the central role of serine protease cascades in regulating insect immune defenses, we focused the remainder of our analysis on a clip-domain serine protease C2 (CLIPC2, AGAP004317) and a serine protease inhibitor 7 (SRPN7, AGAP007693) that were specifically upregulated in the parasite-infected, aseptic mosquito midgut. We showed that SRPN7 negatively and CLIPC2 positively regulate the anti-Plasmodium defense, independently of the midgut-associated bacteria. Co-silencing assays suggested that these two genes may function together in a signaling cascade. Neither gene was regulated, nor modulated, by infection with the rodent malaria parasite Plasmodium berghei, suggesting that SRPN7 and CLIPC2 are components of a defense system with preferential activity towards P. falciparum. Further analysis using RNA interference determined that these genes do not regulate the anti-Plasmodium defense mediated by the IMD pathway, and both factors act as agonists of the endogenous midgut microbiota, further demonstrating the lack of functional relatedness between these genes and the bacteria-dependent activation of the IMD pathway. This is the first study confirming the existence of a bacteria-independent, anti-P. falciparum defense. Aseptic and septic midguts and carcasses from P. falciparum-infected A. gambiae vs aseptic and septic midguts and carcasses from uninfected, blood-fed A. gambiae. 3 biological replicates and 1 pseudo-replicate per each array.

Project description:Background: Mosquito midgut is an important target for the host parasite interaction studies as this major immune organ plays a role in parasite growth, maturation and vector competence. Most important rural malaria vector of India is Proteomic approaches coupled with bioinformatics analysis have been used to study expression of functional proteins/enzymes of An. culicifacies susceptible and refractory species midgut in order to understand the mechanism of refractoriness that may help in contributing to unravel the host pathogen interactions Methodology/Principle findings: Basic shot gun proteomics approaches along with iTRAQ based differential studies followed by LC/MS/MS were employed to study the proteome of sugar fed An. culicifacies midgut. Further bioinformatics analysis was carried out using Gene ontology, SMART analysis, CELLO algorithm etc. Here proteins identified with iTRAQ Ratio more than 1.5 was considered up regulated and less than 0.67 were considered down regulated. Ratio from 1.5 – 0.67 were considered as moderate to no change. Using these criteria we observed total 80 proteins in An. culicifacies midgut and when compared susceptible species against refractory species, total 30 proteins were found to be downregulated. 3 proteins were found to be upregulated in refractory species Conclusion/Significance: Based on the present data a significant increase in number of proteins in midgut of refractory An. culicifacies species B were found that may conclude that these proteins may be responsible for the inhibiting parasite growth and linked to the melanization of oocysts or parasite lysis mechanisms in natural populations of refractory mosquito. Hence the progress of our studies at protein level suggests that these identified annotated putative proteins/enzymes may help to explore natural vector-parasite systems and reveal valuable insights into the mechanism of refractoriness which in turn further useful for bringing of novel strategies for control of malaria.

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Dual RNA-seq of parasite and host reveals gene expression dynamics during filarial worm-mosquito interactions

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