C. elegans is exposed to many different bacteria in its environment, and must distinguish pathogenic from nutritious bacterial food sources. Here, we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance, both in the treated animals and in four subsequent generations of progeny. The RNA interference and piRNA pathways, the germline, and the ASI neuron are required for bacterial small RNA-induced avoidance behavior and transgenerational inheritance. A single non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. A natural microbiome Pseudomonas isolate, GRb0427, can induce avoidance via its small RNAs, and the wild C. elegans strain JU1580 responds similarly to bacterial sRNA. Our results suggest that this ncRNA-dependent mechanism evolved to survey the worm's microbial environment, use this information to make appropriate behavioral decisions, and pass this information on to its progeny.Small RNAs from P. aeruginosa induce species-specific avoidance C. elegans is surrounded by and consumes bacteria as its primary nutrient source. Its natural habitat contains many different bacterial species; about a third of these are in the Pseudomonas family (Samuel et al., 2016), which can be either beneficial or detrimental to the worms. Despite their natural attraction to pathogenic Pseudomonas aeruginosa (PA14), C. elegans can learn to avoid this pathogen after becoming ill (Zhang et al., 2005). Recently, we discovered that worms epigenetically pass on to their progeny this learned avoidance of PA14

In the pathogen Pseudomonas aeruginosa, LasR is a quorum sensing (QS) master regulator that senses the concentration of secreted autoinducers as a proxy for bacterial cell density. Counterintuitively, previous studies showed that saturating amounts of the LasR ligand, 3OC12-HSL, fail to induce the full LasR regulon in low-density liquid cultures. Here we demonstrate that surface association, which is necessary for many of the same group behaviors as QS, promotes stronger QS responses. We show that lasR is upregulated upon surface association, and that surface-associated bacteria induce LasR targets more strongly in response to autoinducer than planktonic cultures. This increased sensitivity may be due to surface-dependent lasR induction initiating a positive feedback loop through the small RNA, Lrs1. The increased sensitivity of surface-associated cells to QS is affected by the type IV pilus (TFP) retraction motors and the minor pilins. The coupling of physical surface responses and chemical QS responses could enable these bacteria to trigger community behaviors more robustly when they are more beneficial.

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surfaceinduced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.

The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

C. elegans is exposed to many different bacteria in its environment, and must distinguish pathogenic from nutritious bacterial food sources. Here, we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance, both in the treated animals and in four subsequent generations of progeny. The RNA interference and piRNA pathways, the germline, and the ASI neuron are required for bacterial small RNA-induced avoidance behavior and transgenerational inheritance. A single non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. A natural microbiome Pseudomonas isolate, GRb0427, can induce avoidance via its small RNAs, and the wild C. elegans strain JU1580 responds similarly to bacterial sRNA. Our results suggest that this ncRNA-dependent mechanism evolved to survey the worm's microbial environment, use this information to make appropriate behavioral decisions, and pass this information on to its progeny.

Surface attachment, an early step in the colonization of multiple host environments, 9 activates the virulence of the human pathogen P. aeruginosa. However, the signaling pathways and 10 downstream toxins specifically induced by surface association to stimulate P. aeruginosa virulence 11 are not fully understood. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites 12 are rapidly induced upon surface association and represent a major class of surface-dependent 13 cytotoxins. AQ cytotoxicity is direct and independent of other AQ functions like quorum sensing or 14 PQS-specific activities like iron sequestration. Furthermore, the regulation of AQ production can 15 explain the surface-dependent virulence regulation of the quorum sensing receptor, LasR, and the 16 pilin-associated candidate mechanosensor, PilY1. PilY1 regulates surface-induced AQ production by 17 repressing the AlgR-AlgZ two-component system. AQs also contribute to the known cytotoxicity of 18 secreted outer membrane vesicles. These findings collectively explain previously mysterious 19 aspects of virulence regulation and provide new avenues for the development of anti-infectives. 20 21 38 LasR is an important component of the complex network of P. aeruginosa quorum sensing (Lee 39 and Zhang, 2015). Quorum sensing (QS) is the process by which bacteria synthesize and secrete 40 1 of 20 Manuscript submitted to eLife autoinducer signaling molecules that accumulate and activate their receptors as a function of 41 bacterial cell density. There are at least three QS systems that have been previously implicated in 42 regulating P. aeruginosa virulence: the las, rhl, and pqs QS systems (Lee and Zhang, 2015). These 43 systems form a complex and interconnected network with extensive regulatory cross-talk (Maura 44 et al., 2016). For example, LasR transcriptionally upregulates the autoinducer synthase enzymes of 45 the rhl and pqs systems (Xiao et al., 2006b; Farrow et al., 2008), which in turn activate numerous 46 downstream factors (Farrow et al., 2008). As a result, identifying the specific contribution of LasR 47 109 PQS while pqsH mutants would have HHQ but no PQS. We found that transient HHQ addition was 110 3 of 20 Manuscript submitted to eLife sufficient to restore WT-levels of virulence to pqsH mutants but not to pqsA mutants (Figure 1-S2), 111 indicating that HHQ is required for surface-induced virulence but PQS is not. 112 Having eliminated PQS-specific roles of AQs such as iron sequestration, we next tested if surface-113 induced virulence requires PqsR activation to achieve high levels of the AQs themselves or if 114 additional PqsR targets (Maura et al., 2016) might be required. Consequently, we replaced the 115 endogenous pqsA promoter with a strong constitutive promoter (P OXB20 ), which we refer to as (P const ). 116 This construct was sufficient to fully restore the virulence of pqsR and pqsH mutants to WT levels 117 Pmid:20978535. 595 16 of 20 Manuscript submitted to eLife Nixon GM, Armstrong DS, Carzino R, Carl...

Pseudomonas aeruginosa is a significant threat in healthcare settings where it deploys a wide host of virulence factors to cause disease. Many virulence-related phenotypes such as pyocyanin production, biofilm formation, and twitching motility have been implicated in causing disease in a number of hosts. In this study, we investigate these three virulence factors in a collection of 22 clinical strains isolated from blood stream infections. Despite the fact that all 22 strains caused disease and came from the same body site of different patients, they show significant variability in assays for each of the three specific phenotypes examined. There was no significant correlation between the strength of the three phenotypes across our collection, suggesting that they can be independently modulated. Furthermore, strains deficient in each of the virulence-associated phenotypes examined could be identified. To understand the genetic basis of this variability we sequenced the genomes of the 22 strains. We found that the majority of genes responsible for pyocyanin production, biofilm formation, and twitching motility were highly conserved among the strains despite their phenotypic variability, suggesting that the phenotypic variability is likely due to regulatory changes. Our findings thus demonstrate that no one lab-assayed phenotype of pyocyanin production, biofilm production, and twitching motility is necessary for a P. aeruginosa strain to cause blood stream infection and that additional factors may be needed to fully predict what strains will lead to specific human diseases.

In vivo tissues have finely controlled hierarchical structure that is often difficult to mimic in vitro. Microfabrication techniques, such as microcontact printing, can be used to reproduce tissue structure in vitro by controlling cell shape and orientation [1]. Several recent results suggest that cellular organization and structure can influence tissue function in engineered tissues [2–4]. For example, using microcontact printing and muscular thin film technology, we recently demonstrated that engineered vascular tissues whose smooth muscle cells possessed more elongated spindle-like geometries, similar to in vivo structure, exhibited more physiological contractile function [5]. In these studies, cells were seeded using traditional imprecise seeding methods. But recent results have shown that cell-cell coupling plays a significant role in functional contractility [6], suggesting that not only cellular geometry, but cell-cell organization, within a tissue is important to reproduce in engineered tissues to mimic in vivo function.