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.

A conserved motif in the membrane proximal C-terminal tail of human muscarinic m1 acetylcholine receptors affects plasma membrane expression.

We investigated the functional role of a conserved motif, F(x)(6)LL, in the membrane proximal C-tail of the human muscarinic M(1) (hM(1)) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence (423)FRDTFRLLL(431). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [(3)H]N-methylscopolamine binding assays. The plasma membrane expression of hM(1) receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM(1) receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L(430) and L(431) play a role in folding hM(1) receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V(46) and L(47), that, when mutated, reduce the plasma membrane expression of hM(1) receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM(1) receptors to achieve a transport-competent state.

Cysteine pairs in the third intracellular loop of the muscarinic m1 acetylcholine receptor play a role in agonist-induced internalization.

We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M(1) (hM(1)) receptor. Using site-directed mutagenesis, several mutant hM(1) receptors were made possessing either a deletion or point mutations within the third intracellular loop domain (252)PETPPGRCCRCC(263). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [(3)H]N-methylscopolamine (pK(D) = approximately 9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC(50) = approximately 5.5) relative to wild-type (pEC(50) = 5.35 +/- 0.05). In contrast, carbachol-induced internalization of mutant hM(1) receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM(1) receptor. Of the hM(1) receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM(1) receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM(1) receptors.