LasR regulates protease IV expression at suboptimal growth temperatures in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa causes debilitating lung infections in people with cystic fibrosis, as well as eye, burn, and wound infections in otherwise immunocompetent individuals. Many of P. aeruginosa 's virulence factors are regulated by environmental changes associated with human infection, such as a change in temperature from ambient to human body temperature. One such virulence factor is protease IV (PIV). Interestingly, piv expression is higher at ambient temperatures (22-28°C) compared to human body temperature (37°C). We found that piv expression was thermoregulated at stationary phase, but not exponential phase, and that piv is thermoregulated at the level of transcription. Protein levels of known transcriptional regulators of piv , the quorum sensing regulator LasR and the gene-silencing histone nucleoid silencing proteins MvaT/MvaU, were not thermoregulated. Using a transcriptional reporter for piv , we show that LasR activates piv expression at stationary phase at 25°C but not 37°C, while MvaT/MvaU are not required for piv thermoregulation. We also identified a las box in the piv promoter, which is important for piv thermoregulation. We propose that LasR directly regulates piv at stationary phase at 25°C but has a negligible impact at 37°C. Here, we show that piv is uniquely regulated by LasR in a temperature-dependent manner. Our findings suggest that the LasRI quorum sensing regulon of P. aeruginosa may not be fully characterized and that growth at non-standard laboratory conditions such as lower temperatures could reveal previously unrecognized quorum sensing regulated genes. IMPORTANCE: Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of causing many different types of infections that are often difficult to treat, such as lung infections in people with cystic fibrosis. Temperature regulates the expression of many virulence factors that contribute to P. aeruginosa 's ability to cause infection, yet our mechanistic understanding of virulence factor thermoregulation is poor. In this study, we show that the virulence factor protease IV is thermoregulated at the level of transcription via temperature-dependent upregulation by the quorum sensing regulator, LasR. Mechanistic studies of virulence factor thermoregulation will expand our understanding of how P. aeruginosa experiences different environments, including the mammalian host. Our work also highlights the importance of growth conditions in studying gene regulation, as it elucidates the regulation of protease IV by LasR, which was previously not well understood.

Social hierarchy reveals thermoregulatory trade-offs in response to repeated stressors.

Coping with stressors can require substantial energetic investment, and when resources are limited, such investment can preclude simultaneous expenditure on other biological processes. Among endotherms, energetic demands of thermoregulation can also be immense, yet our understanding of whether a stress response is sufficient to induce changes in thermoregulatory investment is limited. Using the black-capped chickadee as a model species, we tested a hypothesis that stress-induced changes in surface temperature (Ts), a well-documented phenomenon across vertebrates, stem from trade-offs between thermoregulation and stress responsiveness. Because social subordination is known to constrain access to resources in this species, we predicted that Ts and dry heat loss of social subordinates, but not social dominants, would fall under stress exposure at low ambient temperatures (Ta), and rise under stress exposure at high Ta, thus permitting a reduction in total energetic expenditure toward thermoregulation. To test our predictions, we exposed four social groups of chickadees to repeated stressors and control conditions across a Ta gradient (n=30 days/treatment/group), whilst remotely monitoring social interactions and Ts Supporting our hypothesis, we show that: (1) social subordinates (n=12), who fed less than social dominants and alone experienced stress-induced mass-loss, displayed significantly larger changes in Ts following stress exposure than social dominants (n=8), and (2) stress-induced changes in Ts significantly increased heat conservation at low Ta and heat dissipation at high Ta among social subordinates alone. These results suggest that chickadees adjust their thermoregulatory strategies during stress exposure when resources are limited by ecologically relevant processes.

Evidence that stress-induced changes in surface temperature serve a thermoregulatory function.

The fact that body temperature can rise or fall following exposure to stressors has been known for nearly two millennia; however, the functional value of this phenomenon remains poorly understood. We tested two competing hypotheses to explain stress-induced changes in temperature, with respect to surface tissues. Under the first hypothesis, changes in surface temperature are a consequence of vasoconstriction that occur to attenuate blood loss in the event of injury and serve no functional purpose per se; defined as the 'haemoprotective hypothesis'. Under the second hypothesis, changes in surface temperature reduce thermoregulatory burdens experienced during activation of a stress response, and thus hold a direct functional value: the 'thermoprotective hypothesis'. To understand whether stress-induced changes in surface temperature have functional consequences, we tested predictions of these two hypotheses by exposing black-capped chickadees (n=20) to rotating stressors across an ecologically relevant ambient temperature gradient, while non-invasively monitoring surface temperature (eye region temperature) using infrared thermography. Our results show that individuals exposed to rotating stressors reduce surface temperature and dry heat loss at low ambient temperature and increase surface temperature and dry heat loss at high ambient temperature, when compared with controls. These results support the thermoprotective hypothesis and suggest that changes in surface temperature following stress exposure have functional consequences and are consistent with an adaptation. Such findings emphasize the importance of the thermal environment in shaping physiological responses to stressors in vertebrates, and in doing so, raise questions about their suitability within the context of a changing climate.

The effect of social group size on feather corticosterone in the co-operatively breeding Smooth-billed Ani (Crotophaga ani): An assay validation and analysis of extreme social living.

Living closely with others can provide a myriad of fitness benefits, from shared territory defense to co-operative resource acquisition. Costs of social aggregation are not absent, however, and likely influence optimal and observed groups' sizes in a social species. Here, we explored optimal group size in a joint-nesting cuckoo species (the Smooth-billed Ani, Crotophaga ani) using endocrine markers of stress physiology (corticosterone, or CORT). Smooth-billed Anis exhibit intense reproductive competition that is exacerbated in atypically large groups. We therefore hypothesized that intra-group competition (measured by social group size) mediates the desirability and physiological cost of social group membership in this species. To test this hypothesis, we captured 47 adult Smooth-billed Anis (31 males, 16 females) during the breeding seasons of 2012-2014 in south-western Puerto Rico, and documented social group sizes. Tail feathers were sampled and used to quantify CORT (pg/mg) in enzyme-linked immunosorbent assays (ELISAs) (n = 50). Our analyses show significant differences in feather-CORT of adults between categorical group sizes, with individuals from atypically large social groups (≥ x + 1SD) having highest mean concentrations (33.319 pg/mg), and individuals from atypically small social groups (≤ x - 1SD) having lowest mean concentrations (8.969 pg/mg). Whether reproductive competition or effort is responsible for elevated CORT in atypically large social groups, however, remains unclear. Our results suggest that living in atypically large groups is physiologically expensive and may represent an evolutionarily unstable strategy. To our knowledge, this is the first study to explore a correlation between stress physiology and group size in a joint-nesting species.

Targeting the Wnt pathway in zebrafish as a screening method to identify novel therapeutic compounds.

Activating mutations in the Wnt signaling pathway account for the initiation of greater than 90% of all colorectal cancers and this pathway has been implicated in numerous other diseases. Therefore, identifying small molecule inhibitors of this pathway is of critical importance towards identifying clinically relevant drugs. Numerous screens have been employed to identify therapeutic reagents, but none have made it to advanced clinical trials, suggesting that traditional screening methods are ineffective at identifying clinically relevant targets. Here, we describe a novel in vivo screen to identify small molecule inhibitors of the Wnt pathway. Specifically, treatment of zebrafish embryos with LiCl inhibits GSK3 kinase function, resulting in hyperactivation of the signaling pathway and an eyeless phenotype at 1 day post fertilization. Using the small molecule XAV939, a known inhibitor of Wnt signaling, we rescued the LiCl induced eyeless phenotype, confirming efficacy of the screen. We next tested our assay with 400 known small molecule kinase inhibitors, none of which should inhibit Wnt signaling below the level of GSK3 based on their known targets. Accordingly, none of these small molecules rescued the eyeless phenotype, which demonstrates the stringency of the assay. However, several of these small molecule kinase inhibitors did generate a non-Wnt phenotype in accordance with the kinase they targeted. Therefore, combining the efficacy, sensitivity, and stringency of this preliminary screen, this model will provide an alternative to the traditional in vitro screen, generating potentially clinical relevant drugs in a rapid and cost-effective way.

Dishevelled2 is a stable protein during early zebrafish development.

Wnt signaling is a major player during development and its misregulation often leads to disease, especially cancer. The negative feedback Wnt regulator homologs, Nkd1 and Nkd2, have been shown to inhibit Wnt signaling during development, and current evidence suggests that Nkds degrade Dvl proteins to antagonize Wnt signaling. Here, we demonstrate that during early zebrafish development Nkd1 does not alter either endogenous or exogenous levels of Dvl2. Furthermore, Dvl2 does not affect the levels of Nkd1. Cumulatively, these results demonstrate that Dvl2 is a ubiquitous and stable protein and that Nkds may not always function to degrade Dvl proteins as a method of inhibiting Wnt signaling.