Glycerol metabolism impacts biofilm phenotypes and virulence in Pseudomonas aeruginosa via the Entner-Doudoroff pathway.

Pseudomonas aeruginosa is a ubiquitous bacterium and a notorious opportunistic pathogen that forms biofilm structures in response to many environmental cues. Biofilm formation includes attachment to surfaces and the production of the exopolysaccharide Pel, which is present in both the PAO1 and PA14 laboratory strains of P. aeruginosa. Biofilms help protect bacterial cells from host defenses and antibiotics and abet infection. The carbon source used by the cells also influences biofilm, but these effects have not been deeply studied. We show here that glycerol, which can be liberated from host surfactants during infection, encourages surface attachment and magnifies colony morphology differences. We find that glycerol kinase is important but not essential for glycerol utilization and relatively unimportant for biofilm behaviors. Among downstream enzymes predicted to take part in glycerol utilization, Edd stood out as being important for glycerol utilization and for enhanced biofilm phenotypes in the presence of glycerol. Thus, gluconeogenesis and catabolism of anabolically produced glucose appear to impact not only the utilization of glycerol but also glycerol-stimulated biofilm phenotypes. Finally, waxworm moth larvae and nematode infection models reveal that interruption of the Entner-Doudoroff pathway, but not abrogation of glycerol phosphorylation, unexpectedly increases P. aeruginosa lethality in both acute and chronic infections, even while stimulating a stronger immune response by Caenorhabditis elegans.IMPORTANCEPseudomonas aeruginosa, the ubiquitous environmental bacterium and human pathogen, forms multicellular communities known as biofilms in response to various stimuli. We find that glycerol, a common carbon source that bacteria can use for energy and biosynthesis, encourages biofilm behaviors such as surface attachment and colony wrinkling by P. aeruginosa. Glycerol can be derived from surfactants that are present in the human lungs, a common infection site. Glycerol-stimulated biofilm phenotypes do not depend on phosphorylation of glycerol but are surprisingly impacted by a glucose breakdown pathway, suggesting that it is glycerol utilization, and not its mere presence or cellular import, that stimulates biofilm phenotypes. Moreover, the same mutations that block glycerol-stimulated biofilm phenotypes also impact P. aeruginosa virulence in both acute and chronic animal models. Notably, a glucose-breakdown mutant (Δedd) counteracts biofilm phenotypes but shows enhanced virulence and stimulates a stronger immune response in Caenorhabditis elegans.

Probiotic effects of Lactococcus lactis and Leuconostoc mesenteroides on stress and longevity in Caenorhabditis elegans.

The short lifespan of Caenorhabditis elegans enables the efficient investigation of probiotic interventions affecting stress and longevity involving the potential therapeutic value of Lactococcus lactis and Leuconostoc mesenteroides isolated from organic basil. The lactic acid bacteria were cultured from the produce collected from a local grocery store in Tulsa, Oklahoma, and then identified through 16S rDNA sequencing and biochemical tests. To dive deep into this analysis for potential probiotic therapy, we used fluorescent reporters that allow us to assess the differential induction of multiple stress pathways such as oxidative stress and the cytoplasmic, endoplasmic reticulum, and the mitochondrial unfolded protein response. This is combined with the classic health span measurements of survival, development, and fecundity, allowing a wide range of organismal observations of the different communities of microbes supported by probiotic supplementation with Lactococcus lactis and Leuconostoc mesenteroides. These strains were initially assessed in relation to the Escherichia coli feeding strain OP50 and the C. elegans microbiome. The supplementation showed a reduction in the median lifespan of the worms colonized within the microbiome. This was unsurprising, as negative results are common when probiotics are introduced into healthy microbiomes. To further assess the supplementation potential of these strains on an unhealthy (undifferentiated) microbiome, the typical axenic C. elegans diet, OP50, was used to simulate this single-species biome. The addition of lactic acid bacteria to OP50 led to a significant improvement in the median and overall survival in simulated biomes, indicating their potential in probiotic therapy. The study analyzed the supplemented cultures in terms of C. elegans' morphology, locomotor behavior, reproduction, and stress responses, revealing unique characteristics and stress response patterns for each group. As the microbiome's influence on the health span gains interest, the study aims to understand the microbiome relationships that result in differential stress resistance and lifespans by supplementing microbiomes with Lactococcus lactis and Leuconostoc mesenteroides isolated from organic basil in C. elegans.

Stress and timing associated with Caenorhabditis elegans immobilization methods.

BACKGROUND: Caenorhabditis elegans is a model organism used to study gene, protein, and cell influence on function and behavior. These studies frequently require C. elegans to be immobilized for imaging or laser ablation experiments. There are a number of known techniques for immobilizing worms, but to our knowledge, there are no comprehensive studies of the various agents in common use today. NEW METHOD: This study determines the relationship between concentration, immobilization time, exposure time, and recovery likelihood for several immobilization agents. The agents used in this study are 1-Phenoxy-2-propanol, levamisole, sodium azide, polystyrene beads, and environmental cold shock. These tests are conducted using a humidified chamber to keep chemical concentrations consistent. Each of these agents is also tested to determine if they exhibit stress-related after effects using the gcs-1, daf-16, hsp-4, hif-1, hsp-16.2, and tmem-135 stress reporters. RESULTS: We present a range of quick mount immobilization and recovery conditions for each agent tested. This study shows that, under controlled conditions, 1-Phenoxy-2-propanol shows significant stress from the daf-16 reporter. While 1-Phenoxy-2-propanol and sodium azide both create stress related after effects with long term recovery in the case of the hsp-16.2 reporter. COMPARISON WITH EXISTING METHODS: This study shows that commonly used concentrations of immobilizing agents are ineffective when evaporation is prevented. CONCLUSIONS: To improve reproducibility of results it is essential to use consistent concentrations of immobilizing agents. It is also critically important to account for stress-related after effects elicited by immobilization agents when designing any experiment.

Unusual regulation of splicing of the cholinergic locus in Caenorhabditis elegans.

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.

Hsp90-dependent assembly of the DBC2/RhoBTB2-Cullin3 E3-ligase complex.

The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes.

Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins.

The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells.

BACKGROUND: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. METHODS: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. RESULTS: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. CONCLUSIONS: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Gambogic acid, a natural product inhibitor of Hsp90.

A high-throughput screening of natural product libraries identified (-)-gambogic acid (1), a component of the exudate of Garcinia harburyi, as a potential Hsp90 inhibitor, in addition to the known Hsp90 inhibitor celastrol (2). Subsequent testing established that 1 inhibited cell proliferation, brought about the degradation of Hsp90 client proteins in cultured cells, and induced the expression of Hsp70 and Hsp90, which are hallmarks of Hsp90 inhibition. Gambogic acid also disrupted the interaction of Hsp90, Hsp70, and Cdc37 with the heme-regulated eIF2α kinase (HRI, an Hsp90-dependent client) and blocked the maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that 1 bound to the N-terminal domain of Hsp90 with a low micromolar Kd, in a manner that was not competitive with the Hsp90 inhibitor geldanamycin (3). Molecular docking experiments supported the posit that 1 binds Hsp90 at a site distinct from Hsp90s ATP binding pocket. The data obtained have firmly established 1 as a novel Hsp90 inhibitor and have provided evidence of a new site that can be targeted for the development of improved Hsp90 inhibitors.

Assays for identification of Hsp90 inhibitors and biochemical methods for discriminating their mechanism of action.

The Hsp90-dependence of many oncogenic proteins has precipitated a great deal of interest in Hsp90 as a drug target, as evidence mounts that Hsp90 inhibitors may be effective chemotherapeutic agents for the treatment of cancer. In addition, Hsp90-inhibitors have shown promise for the treatment of neurodegenerative diseases characterized by the accumulation of toxic denatured protein aggregates. The development of assays for the identification of novel Hsp90 inhibitors began in earnest when it became apparent that the Hsp90 inhibitors available at the time had less than ideal pharmacological properties. This review summarizes what is known about Hsp90's structure and function, its ATPase cycle, its interactions with co-chaperones and clients, and the effect of Hsp90-inhibitors on these processes. It further summarizes various high throughput assays (and secondary confirmatory assays) developed to identify new Hsp90 inhibitors from chemical libraries based on the inhibitors ability to: inhibit Hsp90's ATPase activity; compete for ligand binding to Hsp90 and its N-terminal ATP-binding domain; inhibit Hsp90-dependent refolding of denatured luciferase; and deplete culture cells of Hsp90-dependnet client protein or induce Hsp70 expression. In addition, in vitro assays are described that help determine the site of inhibitor binding to Hsp90 (N- or C-terminal domain) and there mechanism of action based on effects of inhibitors on Hsp90/ co-chaperone and client interactions, and Hsp90 conformation characterized by proteolytic fingerprinting.