Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have demonstrated that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density within hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) suppress expression of key immune genes. We then functionally link immune suppression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These ‘biotypes’ have distinct patterns of heritable symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no immune gene suppression from Regiella and host symbionts at lower densities. We then generated F1 hybrids between two biotypes and found that symbiont density and immune suppression are both intermediate in hybrids. We suggest that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. The specific immune mechanisms suppressed by Regiella have been previously demonstrated to combat pathogens in aphids, and thus this work highlights the immune system’s complex dual role in interacting with both beneficial and harmful microbes.Author SummaryHeritable microbes are found in most insects including agriculturally and medically relevant pests. Explaining the variation in the distribution and abundance of symbionts in natural populations is critical to understanding these interactions. This work contributes to our mechanistic understanding of an important model of host-microbe symbiosis and suggests more broadly that variation in insect immune responses plays a role in intraspecific variation in host-symbiont interactions. Our work also suggests that antagonistic coevolution can play a role in host-microbe interactions even when microbes are transmitted vertically and provide a clear benefit to their hosts.

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiontRegiella insecticola(but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These ‘biotypes’ have distinct patterns of symbiont infections: for example, aphids from theTrifoliumbiotype are strongly associated withRegiella. Using RNAseq, we compare patterns of gene expression in response toRegiellain aphid genotypes from multiple biotypes, and we show thatTrifoliumaphids experience no downregulation of immune gene expression while hostingRegiellaand harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system,Regiellasymbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence ofRegiellahave been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system’s complex dual role in interacting with both beneficial and harmful microbes.

The respiratory-gated noncontrast SPACE sequence provided excellent imaging characteristics of the central veins in healthy subjects with promising diagnostic accuracy in patients with central venous pathology.

word count: 229 18 Text word count: 3540 19 20 21 22 23 24 25 26 27 28 2 ABSTRACT 29Evolutionary arms races among organisms are broadly prevalent and bacteria have evolved 30 defensive strategies against various attackers. A common microbial aggression mechanism is the 31 Type VI Secretion System (T6SS), a contact-dependent bacterial weapon used to deliver toxic 32 effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins 33 that neutralize effectors. However, less is known about mechanisms that allow non-sibling bacteria 34 to respond to external cues and survive T6SS attacks independently of immunity proteins. In this 35 study, we show that resistance to T6SS attacks is promoted by a genetically controlled response to 36 exogenous glucose. We observe that multiple human Escherichia coli commensal strains lacking 37 immunity proteins are sensitive to T6SS attacks from pandemic Vibrio cholerae on nutrient-rich 38 media. By contrast, E. coli cells become resistant to attacks when co-cultured on the same media 39 with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find 40 that cAMP receptor protein (CRP), which alters expression of hundreds of genes in response to 41 glucose, controls resistance to T6SS attacks in E. coli cells. Consistent with the observed resistance 42 on media with glucose, an E. coli crp disruption mutant survives significantly better against V. 43 cholerae T6SS attacks even in the absence of glucose. Finally, we also show that resistance to 44 T6SS attacks depends on the pH of the medium and varies based on the target and killer strains. 45 IMPORTANCE 46Many Gram-negative bacteria, including important pathogens, encode T6SS genes to deliver toxic 47 effectors and eliminate competitors. Our results uncover a novel defense mechanism against T6SS 48 attacks that is triggered by an external stimulus and mediated by a metabolic response in non-kin 49 target cells. In microbiomes such as those in gastrointestinal tracts where T6SS activity is known 50 to occur, signaling by metabolites like glucose may affect the efficacy of T6SS attacks and alter 51 3 microbial community composition. Our findings could have vast implications for microbial 52 interactions during pathogen colonization of hosts and survival of bacterial cells in environmental 53 communities. Furthermore, the glucose-mediated resistance observed here might provide a novel 54 example of an evolutionary arms race between killer T6SS cells and target bacteria. 55 INTRODUCTION 56 Vibrio cholerae is the waterborne enteric pathogen that causes serious, often fatal cholera diarrheal 57 disease when ingested by humans. This ubiquitous microbe is found in dense polymicrobial marine 58 communities on chitinous surfaces and in animal reservoirs like fish or zooplankton (1-3). To 59 compete with other cells in densely-populated microbial environments, V. cholerae employs a 60 harpoon-like structure called the Type VI Secretion System (T6SS) (4-7). The T6SS pu...