Summary Borrelia burgdorferi (Bb) adapts to its arthropod and mammalian hosts by altering its transcriptional and antigenic profiles in response to

SummaryOuter surface lipoprotein (Osp) C is a virulence factor required for transmission of the Lyme disease agent, Borrelia burgdorferi. We have constructed an inducible promoter system to study the function and regulation of OspC by integrating regulatory elements from the Escherichia coli lac operon into the B. burgdorferi genome. An inducible promoter (flacp) was constructed by inserting a synthetic lac operator sequence between the transcriptional start site and the ribosomal binding site of the B. burgdorferi flgB promoter; flacp was then used to replace the native ospC and rpoS promoters in B. burgdorferi derivatives that constitutively express the E. coli Lac repressor protein (LacI). In vitro, the expression of ospC and rpoS from flacp was dependent on the inducer isopropyl b-D-thiogalactopyranoside and was unaffected by temperature or pH, conditions commonly used to mimic different aspects of the B. burgdorferi life cycle. Our results suggest that OspC is essential immediately upon injection into a mouse and OspC expression must be maintained during the early stages of infection. In addition, the mouse infectivity experiment indicates that this system can be used to regulate B. burgdorferi genes in vivo, within the context of an experimental tickmouse infectious cycle. RpoS is an alternative sigma factor that is required for ospC transcription. However, the role of other temperature-dependent factors has not previously been addressed. Our results with the inducible rpoS strain demonstrate that RpoS alone is sufficient to activate OspC expression, even at 23°C. This is the first functional inducible promoter system developed for use in B. burgdorferi and, for the first time, will provide researchers with the ability to artificially regulate the expression of genes in this pathogenic spirochaete.

Summary The heat shock transcription factor (HSF) is a conserved regulator of heat shock-inducible gene expression. Organismal roles for HSF in physiological processes such as development, aging, and immunity have been defined largely through studies of the single C. elegans HSF homolog, hsf-1. However, the molecular and cell biological properties of hsf-1 in C. elegans are incompletely understood. We generated animals expressing physiological levels of an HSF-1::GFP fusion protein and examined its function, localization, and regulation in vivo. HSF-1::GFP was functional as measured by its ability to rescue phenotypes associated with two hsf-1 mutant alleles. Rescue of hsf-1 stress, aging, and development phenotypes was abolished in a DNA-binding-deficient mutant, demonstrating that the transcriptional targets of hsf-1 are critical to its function even in the absence of stress. Under non-stress conditions, HSF-1::GFP was found primarily in the nucleus. Following heat shock, HSF-1::GFP rapidly and reversibly redistributed into dynamic, sub-nuclear structures that share many properties with human nuclear stress granules, including colocalization with markers of active transcription. Rapid formation of HSF-1 stress granules required HSF-1 DNA binding activity and the threshold for stress granule formation was altered by growth temperature. HSF-1 stress granule formation was not induced by inhibition of IGF signaling, a pathway previously suggested to function upstream of hsf-1. Our findings suggest that development, stress, and aging pathways may regulate HSF-1 function in distinct ways, and that HSF-1 nuclear stress granule formation is an evolutionarily conserved aspect of HSF-1 regulation in vivo.

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.