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Research Directions

It has become increasingly clear that many animals have close, often intimate, relationships with bacteria and viruses. These associations can have profound impacts on host biology. We are a collaborative research group studying insect-microbe interactions, focusing on endosymbiotic bacteria that live inside host cells. Wolbachia bacteria are the most common known endosymbionts, infecting over half of insect species. My prior work has focused on understanding the genetic, mechanistic, and evolutionary underpinnings of traits that govern Wolbachia’s widespread prevalence. Specifically, many Wolbachia manipulate their host’s reproduction in several ways to encourage their spread across generations. Among these, cytoplasmic incompatibility (CI) is the most common, causing embryos to die when Wolbachia-bearing males mate with uninfected females. Females with Wolbachia remain compatible (right). The Shropshire lab aims to deeply understand the genetic, molecular, and mechanistic basis of this powerful bacterial adaptation and Wolbachia‘s basic biology more broadly. Below, we describe a few areas of specific interest and our recent results.

Image: Cytoplasmic incompatibility crossing relationship.

Genetic basis of cytoplasmic incompatibility (CI) is governed by phage genes cifA and cifB.

What is the genetic basis of bacterial reproductive manipulation?

Strictly maternally transmitted endosymbionts are not often genetically manipulable. This makes them a challenging system for genetic studies linking genotypes to phenotypes. However, ‘omic analyses opened the door for investigating associations between gene presence/absence among diverse species and looking for covariance with phenotypes of interest. Using this approach, we determined candidate genes for Wolbachia-induced CI. With collaborators, we tested these genes by leveraging the powerful Drosophila melanogaster model to express Wolbachia genes in flies that did not have Wolbachia (transgenics). We discovered that two genes that we called CI factors A and B (cifA and cifB) caused CI when expressed in testes (LePage et al. 2017 Nature), that cifA expression in females rescued Wolbachia-induced CI (Shropshire et al. 2018 PNAS), and that we could completely recapitulate CI-induced embryonic death and rescue using transgenes alone (Shropshire et al. 2019 PLOS Genetics). The most exciting part? The genes that cause CI and rescue are not technically Wolbachia genes. Instead, they are encoded within Wolbachia‘s prophage. Thus, viral genes directly control arthropod reproduction.

Image: Genetic basis of cytoplasmic incompatibility (CI) is governed by phage genes cifA and cifB.

Discovering the genetic basis of cytoplasmic incompatibility

How do manipulative genes work to modify host biology?

Transgenic work in Drosophila enabled the discovery of the genes that caused CI, but how the proteins contributed to CI remained largely unknown. With collaborators, we used a set of mutagenesis assays to discover numerous amino acids essential for CI and rescue function (Shropshire et al. 2020, PLOS Pathogens). This work revealed that some CifA mutations had comparable impacts on both CI and rescue, suggesting a common mechanism in CifA’s role in the two phenotypes. We would then leverage these mutations and recombinant proteins purified from an E. coli expression system to determine that CifA has DNase and RNase activity and CifB has DNase activity (Kaur et al. 2022 BioRxiv). This nuclease function translates to spermatid DNA damage, which may represent one of the earliest CI-associated abnormalities. You can read more about our understanding of Cif protein functions in Shropshire et al. 2020 eLife.

Image: summary of cytoplasmic incompatibility factor (Cif) protein functions and impacts on sperm DNA integrity.

What causes phenotypic diversity of bacterial reproductive manipulation?

The CI proteins are considerably diverse, spanning at least five phylogenetic clades we call Types. Early genetic and mechanistic work focused on the Cif proteins of wMel Wolbachia, which belong to the Type 1 clade. Leveraging transgenic expression assays, collaborators and I determined that Type 2 Cifs can also contribute to CI-like phenotypes and that divergent Cifs display incompatibilities (Shropshire et al. 2021, Genetics). These results largely supported the hypothesis that CI gene variation would underlay variation in WolbachiaWolbachia incompatibilities, with some caveats.

Additionally, while CI sometimes manifests as complete embryonic death, this is more the exception than the rule. Instead, CI ranges from near insignificant to complete reductions in hatching, varying by host genotype, Wolbachia genotype, and numerous biotic and abiotic factors. For instance, young males (Shropshire et al. 2021, mBio) and males born from older unmated females (Layton et al. Shropshire 2019, mBio) cause stronger CI. The underlying cause of CI strength variation is largely unknown, but we have identified patterns of CI-strength covariance with Wolbachia density and cif-gene expression in the testes.

Image: Cytoplasmic incompatibility factor (Cif) evolution and plausible underpinnings of CI-strength variation.