Inducing novel endosymbioses by implanting bacteria in fungi.

Endosymbioses have profoundly impacted the evolution of life and continue to shape the ecology of a wide range of species. They give rise to new combinations of biochemical capabilities that promote innovation and diversification1,2. Despite the many examples of known endosymbioses across the tree of life, their de novo emergence is rare and challenging to uncover in retrospect3-5. Here we implant bacteria into the filamentous fungus Rhizopus microsporus to follow the fate of artificially induced endosymbioses. Whereas Escherichia coli implanted into the cytosol induced septum formation, effectively halting endosymbiogenesis, Mycetohabitans rhizoxinica was transmitted vertically to the progeny at a low frequency. Continuous positive selection on endosymbiosis mitigated initial fitness constraints by several orders of magnitude upon adaptive evolution. Phenotypic changes were underscored by the accumulation of mutations in the host as the system stabilized. The bacterium produced rhizoxin congeners in its new host, demonstrating the transfer of a metabolic function through induced endosymbiosis. Single-cell implantation thus provides a powerful experimental approach to study critical events at the onset of endosymbiogenesis and opens opportunities for synthetic approaches towards designing endosymbioses with desired traits.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Engineering Endosymbiotic Growth of E. coli in Mammalian Cells.

Endosymbioses are cellular mergers in which one cell lives within another cell and have led to major evolutionary transitions, most prominently to eukaryogenesis. Generation of synthetic endosymbioses aims to provide a defined starting point for studying fundamental processes in emerging endosymbiotic systems and enable the engineering of cells with novel properties. Here, we tested the potential of different bacteria for artificial endosymbiosis in mammalian cells. To this end, we adopted the fluidic force microscopy technology to inject diverse bacteria directly into the cytosol of HeLa cells and examined the endosymbiont-host interactions by real-time fluorescence microscopy. Among them, Escherichia coli grew exponentially within the cytoplasm, however, at a faster pace than its host cell. To slow down the intracellular growth of E. coli, we introduced auxotrophies in E. coli and demonstrated that the intracellular growth rate can be reduced by limiting the uptake of aromatic amino acids. In consequence, the survival of the endosymbiont-host pair was prolonged. The presented experimental framework enables studying endosymbiotic candidate systems at high temporal resolution and at the single cell level. Our work represents a starting point for engineering a stable, vertically inherited endosymbiosis.