Wolbachia and virus alter the host transcriptome at the interface of nucleotide metabolism pathways

Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for Dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.


Host response to virus infection varies depending on time and Wolbachia colonization
We identified 157 genes that were significantly differentially expressed due to virus infection     (Table 1, Supplemental Tables S6 and S7). These genes with interactive effects at the 206 level of splicing were also significantly differentially expressed due to either Wolbachia or virus alone. These 34 differentially spliced genes include a range of predicted functions including 208 transcription and translation (eEF2, MED26, and da), cytoskeletal organization (sickie, CAP, Eb1, 209 hts, and Klp10A), nucleotide metabolic processes (Pde11), and immune and stress responses 210 (Irc and cert), amongst others (Table 1, Supplemental Tables S6 and S7).

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Next, we clustered all infection responsive genes (either at the level of gene expression and/or 213 isoform usage) to determine how interconnected the Wolbachia-and virus-responsive genes sets 214 are. Each gene was classified as either "Wolbachia-responsive", "virus-responsive", "interactionbut non-interactively (for example, differentially expressed due to Wolbachia colonization, and 217 differential isoform usage due to SINV infection). We identified one core network that includes 218 genes across all responses, with numerous connections between Wolbachia-responsive, virus-219 responsive, and interactive response genes ( Figure 5). This clustering revealed that metabolic 220 processes are the most interconnected between the different responses, particularly de novo 221 nucleotide synthesis. Indeed, we identified numerous GO Processes that were significantly 222 enriched in the joint network, all of which were metabolic in nature (Supplemental Table S8).

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Enrichments included amino acid metabolic processes, purine biosynthesis, and other small 224 molecule metabolic processes.  Figure 5). In general, the purine synthesis pathway is strongly downregulated due to virus 235 ( Figure 6B), and the pyrimidine synthesis pathway is strongly downregulated due to Wolbachia 236 (including upregulation of a suppressor, su(r))( Figure 6C). Interestingly, there are a few genes 237 that differentially respond to Wolbachia and virus, such as prat2. prat2 is a gene involved in the

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In both Wolbachia-colonized and Wolbachia-free flies, prat2 knockdown was effective, resulting 251 in prat2 mRNA levels being reduced to 24.3% and 39.6% of the sibling controls, respectively 252 ( Figure 6D). There was no significant difference in prat2 mRNA levels between Wolbachia-    Wolbachia. This deeper look into the association allowed us to more efficiently overlay the 278 changes that occur due to virus, and identify areas of overlapping effects, regardless of whether 279 or not they were combinatorial.

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One of the major findings across our analyses is the significant amount of differential isoform

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The effects of Wolbachia on host metabolism are arguably underexplored (Newton and Rice, 298 2020), which is surprising given that Wolbachia must acquire all nutrients from the host, encodes 299 for a select number of its own metabolic pathways, and encodes for a variety of transporters that 300 would allow for Wolbachia to import specific metabolites (e.x., amino acids) (Wu et al., 2004).

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We next identified the changes in gene expression and isoform usage due to the presence of 303 virus. The virus-responsive network contained fewer cellular processes than did the Wolbachia-304 responsive network: the response to virus mainly affected the expression of endomembrane 305 system associated genes, and metabolic pathways. It is notable that these metabolic pathways