Competitive Exclusion of Phytopathogenic Serratia marcescens from Squash Bug Vectors by the Gut Endosymbiont Caballeronia.

Many insects harbor microbial symbiotic partners that offer protection against pathogens, parasitoids, and other natural enemies. Mounting evidence suggests that these symbiotic microbes can play key roles in determining infection outcomes in insect vectors, making them important players in the quest to develop novel vector control strategies. Using the squash bug Anasa tristis, we investigated how the presence of Caballeronia symbionts affected the persistence and intensity of phytopathogenic Serratia marcescens within the insect vector. We reared insects aposymbiotically and with different Caballeronia isolates, infected them with S. marcescens, and then sampled the insects periodically to assess the intensity and persistence of pathogen infection. Squash bugs harboring Caballeronia consistently had much lower-intensity infections and cleared S. marcescens significantly faster than their aposymbiotic counterparts. These patterns held even when we reversed the timing of exposure to symbiont and pathogen. Taken together, these results indicate that Caballeronia symbionts play an essential role in S. marcescens infection outcomes in squash bugs and could be used to alter vector competence to enhance agricultural productivity in the future. IMPORTANCE Insect-microbe symbioses have repeatedly been shown to profoundly impact an insect's ability to vector pathogens to other hosts. The use of symbiotic microbes to control insect vector populations is of growing interest in agricultural settings. Our study examines how symbiotic microbes affect the dynamics of a plant pathogen infection within the squash bug vector Anasa tristis, a well-documented pest of squash and other cucurbit plants and a vector of Serratia marcescens, the causative agent of cucurbit yellow vine disease. We provide evidence that the symbiont Caballeronia prevents successful, long-term establishment of S. marcescens in the squash bug. These findings give us insight into symbiont-pathogen dynamics within the squash bug that could ultimately determine its ability to transmit pathogens and be leveraged to interrupt disease transmission in this system.

Novel Chlamydiae and Amoebophilus endosymbionts are prevalent in wild isolates of the model social amoeba Dictyostelium discoideum.

Amoebae interact with bacteria in multifaceted ways. Amoeba predation can serve as a selective pressure for the development of bacterial virulence traits. Bacteria may also adapt to life inside amoebae, resulting in symbiotic relationships. Indeed, particular lineages of obligate bacterial endosymbionts have been found in different amoebae. Here, we screened an extensive collection of Dictyostelium discoideum wild isolates for the presence of these bacterial symbionts using endosymbiont specific PCR primers. We find that these symbionts are surprisingly common, identified in 42% of screened isolates (N = 730). Members of the Chlamydiae phylum are particularly prevalent, occurring in 27% of the amoeba isolated. They are novel and phylogenetically distinct from other Chlamydiae. We also found Amoebophilus symbionts in 8% of screened isolates (N = 730). Antibiotic-cured amoebae behave similarly to their Chlamydiae or Amoebophilus-infected counterparts, suggesting that these endosymbionts do not significantly impact host fitness, at least in the laboratory. We found several natural isolates were co-infected with multiple endosymbionts, with no obvious fitness effect of co-infection under laboratory conditions. The high prevalence and novelty of amoeba endosymbiont clades in the model organism D. discoideum opens the door to future research on the significance and mechanisms of amoeba-symbiont interactions.

Endosymbiotic adaptations in three new bacterial species associated with Dictyostelium discoideum: Paraburkholderia agricolaris sp. nov., Paraburkholderia hayleyella sp. nov., and Paraburkholderia bonniea sp. nov.

Here we give names to three new species of Paraburkholderia that can remain in symbiosis indefinitely in the spores of a soil dwelling eukaryote, Dictyostelium discoideum. The new species P. agricolaris sp. nov., P. hayleyella sp. nov., and P. bonniea sp. nov. are widespread across the eastern USA and were isolated as internal symbionts of wild-collected D. discoideum. We describe these sp. nov. using several approaches. Evidence that they are each a distinct new species comes from their phylogenetic position, average nucleotide identity, genome-genome distance, carbon usage, reduced length, cooler optimal growth temperature, metabolic tests, and their previously described ability to invade D. discoideum amoebae and form a symbiotic relationship. All three of these new species facilitate the prolonged carriage of food bacteria by D. discoideum, though they themselves are not food. Further studies of the interactions of these three new species with D. discoideum should be fruitful for understanding the ecology and evolution of symbioses.

Paraburkholderia Symbionts Display Variable Infection Patterns That Are Not Predictive of Amoeba Host Outcomes.

Symbiotic interactions exist within a parasitism to mutualism continuum that is influenced, among others, by genes and context. Dynamics of intracellular invasion, replication, and prevalence may underscore both host survivability and symbiont stability. More infectious symbionts might exert higher corresponding costs to hosts, which could ultimately disadvantage both partners. Here, we quantify infection patterns of diverse Paraburkholderia symbiont genotypes in their amoeba host Dictyostelium discoideum and probe the relationship between these patterns and host outcomes. We exposed D. discoideum to thirteen strains of Paraburkholderia each belonging to one of the three symbiont species found to naturally infect D. discoideum: Paraburkholderia agricolaris, Paraburkholderia hayleyella, and Paraburkholderia bonniea. We quantified the infection prevalence and intracellular density of fluorescently labeled symbionts along with the final host population size using flow cytometry and confocal microscopy. We find that infection phenotypes vary across symbiont strains. Symbionts belonging to the same species generally display similar infection patterns but are interestingly distinct when it comes to host outcomes. This results in final infection loads that do not strongly correlate to final host outcomes, suggesting other genetic factors that are not a direct cause or consequence of symbiont abundance impact host fitness.

Intracellular Burkholderia Symbionts induce extracellular secondary infections; driving diverse host outcomes that vary by genotype and environment.

Symbiotic associations impact and are impacted by their surrounding ecosystem. The association between Burkholderia bacteria and the soil amoeba Dictyostelium discoideum is a tractable model to unravel the biology underlying symbiont-endowed phenotypes and their impacts. Several Burkholderia species stably associate with D. discoideum and typically reduce host fitness in food-rich environments while increasing fitness in food-scarce environments. Burkholderia symbionts are themselves inedible to their hosts but induce co-infections with secondary bacteria that can serve as a food source. Thus, Burkholderia hosts are "farmers" that carry food bacteria to new environments, providing a benefit when food is scarce. We examined the ability of specific Burkholderia genotypes to induce secondary co-infections and assessed host fitness under a range of co-infection conditions and environmental contexts. Although all Burkholderia symbionts intracellularly infected Dictyostelium, we found that co-infections are predominantly extracellular, suggesting that farming benefits are derived from extracellular infection of host structures. Furthermore, levels of secondary infection are linked to conditional host fitness; B. agricolaris infected hosts have the highest level of co-infection and have the highest fitness in food-scarce environments. This study illuminates the phenomenon of co-infection induction across Dictyostelium associated Burkholderia species and exemplifies the contextual complexity of these associations.

The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence, species, genetic and phenotypic diversity.

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia-Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.

Symbiont location, host fitness, and possible coadaptation in a symbiosis between social amoebae and bacteria.

Recent symbioses, particularly facultative ones, are well suited for unravelling the evolutionary give and take between partners. Here we look at variation in natural isolates of the social amoeba Dictyostelium discoideum and their relationships with bacterial symbionts, Burkholderia hayleyella and Burkholderia agricolaris. Only about a third of field-collected amoebae carry a symbiont. We cured and cross-infected amoebae hosts with different symbiont association histories and then compared host responses to each symbiont type. Before curing, field-collected clones did not vary significantly in overall fitness, but infected hosts produced morphologically different multicellular structures. After curing and reinfecting, host fitness declined. However, natural B. hayleyella hosts suffered fewer fitness costs when reinfected with B. hayleyella, indicating that they have evolved mechanisms to tolerate their symbiont. Our work suggests that amoebae hosts have evolved mechanisms to tolerate specific acquired symbionts; exploring host-symbiont relationships that vary within species may provide further insights into disease dynamics.

A dominant-negative mutant inhibits multiple prion variants through a common mechanism.

Prions adopt alternative, self-replicating protein conformations and thereby determine novel phenotypes that are often irreversible. Nevertheless, dominant-negative prion mutants can revert phenotypes associated with some conformations. These observations suggest that, while intervention is possible, distinct inhibitors must be developed to overcome the conformational plasticity of prions. To understand the basis of this specificity, we determined the impact of the G58D mutant of the Sup35 prion on three of its conformational variants, which form amyloids in S. cerevisiae. G58D had been previously proposed to have unique effects on these variants, but our studies suggest a common mechanism. All variants, including those reported to be resistant, are inhibited by G58D but at distinct doses. G58D lowers the kinetic stability of the associated amyloid, enhancing its fragmentation by molecular chaperones, promoting Sup35 resolubilization, and leading to amyloid clearance particularly in daughter cells. Reducing the availability or activity of the chaperone Hsp104, even transiently, reverses curing. Thus, the specificity of inhibition is determined by the sensitivity of variants to the mutant dosage rather than mode of action, challenging the view that a unique inhibitor must be developed to combat each variant.

Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria.

Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

In the social amoeba Dictyostelium discoideum, density, not farming status, determines predatory success on unpalatable Escherichia coli.

BACKGROUND: The social amoeba Dictyostelium discoideum interacts with bacteria in a variety of ways. It is a predator of bacteria, can be infected or harmed by bacteria, and can form symbiotic associations with bacteria. Some clones of D. discoideum function as primitive farmers because they carry bacteria through the normally sterile D. discoideum social stage, then release them after dispersal so the bacteria can proliferate and be harvested. Some farmer-associated bacteria produce small molecules that promote host farmer growth but inhibit the growth of non-farmer competitors. To test whether the farmers' tolerance is specific or extends to other growth inhibitory bacteria, we tested whether farmer and non-farmer amoebae are differentially affected by E. coli strains of varying pathogenicity. Because the numbers of each organism may influence the outcome of amoeba-bacteria interactions, we also examined the influence of amoeba and bacteria density on the ability of D. discoideum to grow and develop on distinct bacterial strains. RESULTS: A subset of E. coli strains did not support amoeba proliferation on rich medium, independent of whether the amoebae were farmers or non-farmers. However, amoebae could proliferate on these strains if amoebae numbers are high relative to bacteria numbers, but again there was no difference in this ability between farmer and non-farmer clones of D. discoideum. CONCLUSIONS: Our results show that farmer and non-farmers did not differ in their abilities to consume novel strains of E. coli, suggesting that farmer resistance to their own carried bacteria does not extend to foreign bacteria. We see that increasing the numbers of bacteria or amoebae increases their respective likelihood of competitive victory over the other, thus showing Allee effects. We hypothesize that higher bacteria numbers may result in higher concentrations of a toxic product or in a reduction of resources critical for amoeba survival, producing an environment inhospitable to amoeba predators. Greater amoeba numbers may counter this growth inhibition, possibly through reducing bacterial numbers via increased predation rates, or by producing something that neutralizes a potentially toxic bacterial product.

Insights into prion biology: integrating a protein misfolding pathway with its cellular environment.

Protein misfolding and assembly into ordered, self-templating aggregates (amyloid) has emerged as a novel mechanism for regulating protein function. For a subclass of amyloidogenic proteins known as prions, this process induces transmissible changes in normal cellular physiology, ranging from neurodegenerative disease in animals and humans to new traits in fungi. The severity and stability of these altered phenotypic states can be attenuated by the conformation or amino-acid sequence of the prion, but in most of these cases, the protein retains the ability to form amyloid in vitro. Thus, our ability to link amyloid formation in vitro with its biological consequences in vivo remains a challenge. In two recent studies, we have begun to address this disconnect by assessing the effects of the cellular environment on traits associated with the misfolding of the yeast prion Sup35. Remarkably, the effects of quality control pathways and of limitations on protein transfer in vivo amplify the effects of even slight differences in the efficiency of Sup35 misfolding, leading to dramatic changes in the associated phenotype. Together, our studies suggest that the interplay between protein misfolding pathways and their cellular context is a crucial contributor to prion biology.

Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation.

Protein misfolding underlies many neurodegenerative diseases, including the transmissible spongiform encephalopathies (prion diseases). Although cells typically recognize and process misfolded proteins, prion proteins evade protective measures by forming stable, self-replicating aggregates. However, coexpression of dominant-negative prion mutants can overcome aggregate accumulation and disease progression through currently unknown pathways. Here we determine the mechanisms by which two mutants of the Saccharomyces cerevisiae Sup35 protein cure the [PSI(+)] prion. We show that both mutants incorporate into wild-type aggregates and alter their physical properties in different ways, diminishing either their assembly rate or their thermodynamic stability. Whereas wild-type aggregates are recalcitrant to cellular intervention, mixed aggregates are disassembled by the molecular chaperone Hsp104. Thus, rather than simply blocking misfolding, dominant-negative prion mutants target multiple events in aggregate biogenesis to enhance their susceptibility to endogenous quality-control pathways.

A size threshold limits prion transmission and establishes phenotypic diversity.

According to the prion hypothesis, atypical phenotypes arise when a prion protein adopts an alternative conformation and persist when that form assembles into self-replicating aggregates. Amyloid formation in vitro provides a model for this protein-misfolding pathway, but the mechanism by which this process interacts with the cellular environment to produce transmissible phenotypes is poorly understood. Using the yeast prion Sup35/[PSI(+)], we found that protein conformation determined the size distribution of aggregates through its interactions with a molecular chaperone. Shifts in this range created variations in aggregate abundance among cells because of a size threshold for transmission, and this heterogeneity, along with aggregate growth and fragmentation, induced age-dependent fluctuations in phenotype. Thus, prion conformations may specify phenotypes as population averages in a dynamic system.