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Brain function depends on synaptic connections between specific neuron types, yet systematic descriptions of synaptic networks and their molecular properties are not readily available. Here, we introduce SBARRO ( S ynaptic B arcode A nalysis by R etrograde R abies Read O ut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its “starter” postsynaptic cell to that cell’s presynaptic partners. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNAs. We use SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, finding that different cell types have presynaptic networks with differences in average size and cell type composition. Patterns of RNA expression suggest that functioning synapses are critical for rabies virus uptake. By tracking individual rabies clones across cells, SBARRO offers new opportunities to map the synaptic organization of neural circuits. Synaptic connections are critical for brain function but are hard to measure systematically. Here, authors present a method which uses rabies virus barcoding and single-cell RNAseq to parallelize monosynaptic network reconstruction from molecularly-profiled single cells.

Intracellular bacterial pathogens secrete effectors to manipulate the host cell environment, create a hospitable niche, and promote infection. While many effectors interact with specific host machinery to perform a single distinct function, some effectors are capable of interaction with multiple host proteins to carry out multiple functions. Rickettsia species are obligate intracellular bacteria that cause vector-borne diseases that constitute an ongoing public health threat. As Rickettsia spp. have small genomes, and thus a limited coding capacity, multifunctional effectors may be an efficient way to manipulate their host environment. However, relatively few secreted effectors have been characterized in the Rickettsia genus and even fewer have been identified as multifunctional effectors.In this work, I demonstrate that the rickettsial secreted effector Sca4 interacts with the host endocytic factor clathrin heavy chain. As previous work showed that Sca4 interacts with the host protein vinculin in mammalian cells, this discovery of the Sca4-clathrin interaction makes Sca4 one of the first multifunctional effectors to be identified in a Rickettsia species. When investigating the potential role of the Sca4-clathrin interaction, I found that clathrin promotes the cell-to-cell spread of R. parkeri in mammalian cells by acting in the recipient cell. However, the Sca4-clathrin interaction was found to be dispensable for efficient cell-to-cell spread. I investigated the role of this interaction in the tick arthropod vector and found that the Sca4-clathrin interaction is necessary for the efficient proliferation of R. parkeri in tick cells. These findings show that knowledge of the complete roles of rickettsial secreted effectors in both arthropod vector and mammalian hosts is needed to fully understand rickettsial pathogenesis.

Intracellular bacterial pathogens deploy secreted effector proteins that manipulate diverse host machinery and pathways to promote infection. Although many effectors carry out a single specific function or interaction, there are a growing number of secreted pathogen effectors capable of interacting with multiple host factors. However, few effectors secreted by obligate intracellular species have been linked to multiple host targets. Here, we investigated the conserved rickettsial secreted effector Sca4, which was previously shown to interact with host vinculin to promote cell-to-cell spread in the model species . We discovered that Sca4 also binds the host cell endocytic factor clathrin heavy chain (CHC, ) via a conserved segment in the Sca4 N-terminus. Ablation of expression or chemical inhibition of endocytosis reduced cell-to-cell spread, indicating that clathrin promotes efficient spread between mammalian cells. This activity was independent of Sca4 and appeared restricted to the recipient host cell, suggesting that the Sca4-clathrin interaction also regulates another aspect of the infectious lifecycle. Indeed, lacking Sca4 or expressing a Sca4 truncation unable to bind clathrin had markedly reduced burdens in tick cells, hinting at a cell-type specific function for the Sca4-clathrin interaction. Sca4 homologs from diverse species also bound clathrin, suggesting that the function of this novel effector-host interaction may be broadly important for rickettsial infection. We conclude that Sca4 has multiple targets during infection and that rickettsiae may manipulate host endocytic machinery to facilitate several stages of their life cycles.

Brain function depends on forming and maintaining connections between neurons of specific types, ensuring neural function while allowing the plasticity necessary for cellular and behavioral dynamics. However, systematic descriptions of how brain cell types organize into synaptic networks and which molecules instruct these relationships are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its \"starter\" postsynaptic cell to that cell's presynaptic partners1. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNA profiles - and thus cell identities and molecular states - of each host cell. We used SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, leveraging a system similar to those used to identify synaptogenic molecules. We found that the molecular identity (cell type/subtype) of the starter cell predicted the number and types of cells that had synapsed onto it. Rabies transmission tended to occur into cells with RNA-expression signatures related to developmental maturation and synaptic transmission. The estimated size of a cell's presynaptic network, relative to that of other cells of the same type, associated with increased expression of Arpp21 and Cdh13. By tracking individual virions and their clonal progeny as they travel among host cells, single-cell, single-virion genomic technologies offer new opportunities to map the synaptic organization of neural circuits in health and disease. Competing Interest Statement The authors have declared no competing interest.

Listeria monocytogenes is a human bacterial pathogen that disseminates through host tissues using a process called cell-to-cell spread. This critical yet understudied virulence strategy resembles a vesicular form of intercellular trafficking that allows L. monocytogenes to move between host cells without escaping the cell. Interestingly, eukaryotic cells can also directly exchange cellular components via intercellular communication pathways (e.g. trans-endocytosis) using cell-cell adhesion, membrane trafficking, and membrane remodeling proteins. Therefore, we hypothesized that L. monocytogenes would hijack these types of host proteins during spread. Using a focused RNAi screen, we identified 22 host genes that are important for L. monocytogenes spread. We then found that caveolins (CAV1 and CAV2) and the membrane sculpting F-BAR protein PACSIN2 promote L. monocytogenes protrusion engulfment during spread, and that PACSIN2 specifically localized to protrusions. Overall, our study demonstrates that host intercellular communication pathways may be co-opted during bacterial spread and that specific trafficking and membrane remodeling proteins promote bacterial protrusion resolution.