Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.DOI: http://dx.doi.org/10.7554/eLife.29397.001

Myxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-type Myxococcus xanthus (DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large "polyploid prophage," Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery of M. xanthus to transfer a toxin that makes the population "addicted" to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (by traA allele swap) to recognize a closely related species, Myxococcus fulvus. As a result, M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms in traA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes. IMPORTANCEThe transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups.

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Background: Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity in atherosclerotic plaques, and among other approaches, has been modeled in vitro by cholesterol loading. Methods: Meta-analysis of scRNA-seq data from VSMC lineage traced cells across five experiments of murine atherosclerosis was performed. In vivo expression profiles were compared to three in vitro datasets of VSMCs loaded with cholesterol and three datasets of polarized macrophages. Results: We identified 24 cell clusters in the meta-analysis of single cells from mouse atherosclerotic lesions with notable heterogeneity across studies, especially for macrophage populations. Trajectory analysis of VSMC lineage positive cells revealed several possible paths of state transitions with one traversing from contractile VSMC to macrophages by way of a proliferative cell cluster. Transcriptome comparisons between in vivo and in vitro states underscored that data from three in vitro cholesterol-treated VSMC experiments did not mirror cell state transitions observed in vivo. However, all in vitro macrophage profiles analyzed (M1, M2, and oxLDL) were more similar to in vivo profiles of macrophages than in vitro VSMCs were to in vivo profiles of VSMCs. oxLDL loaded macrophages showed the most similarity to in vivo states. In contrast to the in vitro data, comparison between mouse and human in vivo data showed many similarities. Conclusions: Identification of the sources of variation across single cell datasets in atherosclerosis will be an important step towards understanding VSMC fate transitions in vivo. Also, we conclude that cholesterol-loading in vitro is insufficient to model the VSMC cell state transitions observed in vivo, which underscores the need to develop better cell models. Mouse models, however, appear to reproduce a number of the features of VSMCs in human plaques.

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension (PAH) severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of PAH cases versus controls, highlighting potentially novel candidate genes involved in PAH development.

Toxoplasma gondii’s tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo. This predilection for neurons suggests that T. gondii’s persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells. IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii’s persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo. By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.

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