Facultative symbiont virulence determines horizontal transmission rate without host specificity in Dictyostelium discoideum social amoebas.

In facultative symbioses, only a fraction of hosts are associated with symbionts. Specific host and symbiont pairings may be the result of host-symbiont coevolution driven by reciprocal selection or priority effects pertaining to which potential symbiont is associated with a host first. Distinguishing between these possibilities is important for understanding the evolutionary forces that affect facultative symbioses. We used the social amoeba, Dictyostelium discoideum, and its symbiont, Paraburkholderia bonniea, to determine whether ongoing coevolution affects which host-symbiont strain pairs naturally cooccur within a facultative symbiosis. Relative to other Paraburkholderia, including another symbiont of D. discoideum, P. bonniea features a reduced genome size that indicates a significant history of coevolution with its host. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness for naturally cooccurring (native) host and symbiont pairings compared to novel pairings. We show for the first time that P. bonniea symbionts can horizontally transmit to new amoeba hosts when hosts aggregate together during the social stage of their life cycle. Here we find evidence for a virulence-transmission trade-off without host specificity. Although symbiont strains were significantly variable in virulence and horizontal transmission rate, hosts and symbionts responded similarly to associations in native and novel pairings. We go on to identify candidate virulence factors in the genomes of P. bonniea strains that may contribute to variation in virulence. We conclude that ongoing coevolution is unlikely for D. discoideum and P. bonniea. The system instead appears to represent a stable facultative symbiosis in which naturally cooccurring P. bonniea host and symbiont pairings are the result of priority effects.

Systematic elucidation of genetic mechanisms underlying cholesterol uptake.

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we substantially improve the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of the genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

Facultative symbiont virulence determines horizontal transmission rate without host strain specificity.

In facultative symbioses, only a fraction of hosts are associated with a symbiont. Understanding why specific host and symbiont strains are associated can inform us of the evolutionary forces affecting facultative symbioses. Possibilities include ongoing host-symbiont coevolution driven by reciprocal selection, or priority effects that are neutral in respect to the host-symbiont interaction. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness estimates for naturally co-occurring (native) host and symbiont combinations compared to nonnative combinations. We used the Dictyostelium discoideum - Paraburkholderia bonniea system to test this hypothesis. P. bonniea features a reduced genome size relative to another Paraburkholderia symbiont of D. discoideum, indicating a significant history of coevolution with its host. Facultative symbionts may experience continued genome reduction if coevolution is ongoing, or their genome size may have reached a stable state if the symbiosis has also stabilized. Our work demonstrates that ongoing coevolution is unlikely for D. discoideum and P. bonniea. The system instead represents a stable facultative symbiosis. Specifically associated host and symbiont strains in this system are the result of priority effects, and presently unassociated hosts are simply uncolonized. We find evidence for a virulence-transmission trade-off without host strain specificity, and identify candidate virulence factors in the genomes of P. bonniea strains that may contribute to variation in benevolence.

Systematic elucidation of genetic mechanisms underlying cholesterol uptake.

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we have substantially improved the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

Reduced and Nonreduced Genomes in Paraburkholderia Symbionts of Social Amoebas.

The social amoeba Dictyostelium discoideum is a predatory soil protist frequently used for studying host-pathogen interactions. A subset of D. discoideum strains isolated from soil persistently carry symbiotic Paraburkholderia, recently formally described as P. agricolaris, P. bonniea, and P. hayleyella. The three facultative symbiont species of D. discoideum present a unique opportunity to study a naturally occurring symbiosis in a laboratory model protist. There is a large difference in genome size between P. agricolaris (8.7 million base pairs [Mbp]) versus P. hayleyella and P. bonniea (4.1 Mbp). We took a comparative genomics approach and compared the three genomes of D. discoideum symbionts to 12 additional Paraburkholderia genomes to test for genome evolution patterns that frequently accompany host adaptation. Overall, P. agricolaris is difficult to distinguish from other Paraburkholderia based on its genome size and content, but the reduced genomes of P. bonniea and P. hayleyella display characteristics indicative of genome streamlining rather than deterioration during adaptation to their protist hosts. In addition, D. discoideum-symbiont genomes have increased secretion system and motility genes that may mediate interactions with their host. Specifically, adjacent BurBor-like type 3 and T6SS-5-like type 6 secretion system operons shared among all three D. discoideum-symbiont genomes may be important for host interaction. Horizontal transfer of these secretion system operons within the amoeba host environment may have contributed to the unique ability of these symbionts to establish and maintain a symbiotic relationship with D. discoideum. IMPORTANCE Protists are a diverse group of typically single cell eukaryotes. Bacteria and archaea that form long-term symbiotic relationships with protists may evolve in additional ways than those in relationships with multicellular eukaryotes such as plants, animals, or fungi. Social amoebas are a predatory soil protist sometimes found with symbiotic bacteria living inside their cells. They present a unique opportunity to explore a naturally occurring symbiosis in a protist frequently used for studying host-pathogen interactions. We show that one amoeba-symbiont species is similar to other related bacteria in genome size and content, while the two reduced-genome-symbiont species show characteristics of genome streamlining rather than deterioration during adaptation to their host. We also identify sets of genes present in all three amoeba-symbiont genomes that are potentially used for host-symbiont interactions. Because the amoeba symbionts are distantly related, the amoeba host environment may be where these genes were shared among symbionts.

Using CRISPR to understand and manipulate gene regulation.

Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics.

The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1.Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.