Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

The objective of this study was to investigate the effect of a phytogenic water additive (PWA) on growth performance and underlying factors involved in pigs fed with low-protein (LP)/high-carbohydrate diets. Forty-eight weaned barrows were allotted to six treatments for 4 weeks: CON-NS, control (CON) diet-no PWA; CON-LS, CON diet-low dose PWA (4 mL/L); CON-HS, CON diet-high dose PWA (8 mL/L); LP-NS, LP diet-no PWA; LP-LS, LP diet-low dose PWA; LP-HS, LP diet-high dose PWA. Relative to CON-NS, pigs fed with CON-HS had increased average daily gain, body weight and serum calcium (Ca) and phosphorous (P) and had decreased mRNA abundance of solute carrier family 7 member 11 and solute carrier family 6 member 19 in jejunum. Compared to LP-NS, pigs fed with LP-HS had increased muscle lean%, decreased muscle fat%, decreased serum Ca and increased serum P. Compared to their NS counterparts, CON-LS, CON-HS, and LP-LS increased the concentration of plasma essential AA and those fed with CON-HS and LP-HS tended to reduce the abundance of the solute carrier family 7 member 1 transcript in skeletal muscle. Thus, PWA improved the performance of weaned pigs fed with protein-adequate diets likely through increased blood essential AA and affected the muscle composition when dietary protein was deficient.

The objective of this study was to assess the effects of low protein diets on feed intake (FI), heat production and markers of FI and thermogenesis regulation in broilers under experimentally induced heat stress. Two-hundred-day-old broiler chicks were weight-matched and assigned into 36 pens (5-6 chicks/pen) followed by assigning them into two treatments (18 pens/treatment): 1) thermoneutral (TN), 2) heat stress (HS). Within each treatment, the pens were randomized to receive two diets (9 pens/diet): 1) normal protein (NP), 2) low protein (LP). The study lasted 6 weeks with 2 weeks of acclimation and 4 weeks of data collection. On week 6, birds were euthanized, and blood and tissue samples collected. All data were analyzed with either GLM or mixed procedure (SPSS). The student’s t-test was used to separate means between NP and LP diets within each treatment. There was no difference in average daily gain between NP and LP diets in the TN group, but LP decreased that during HS (P< 0.05). In TN condition, LP tended to increase the average daily feed intake (ADFI) compared to NP (P< 0.1), while during HS, LP was not different from NP in terms of ADFI (P>0.05). In TN condition, LP had a higher thermal radiation than NP, but LP had less thermal radiation than NP during HS (P< 0.05). In support of ADFI data, LP had a greater ghrelin transcript in the duodenum than NP in TN condition (P< 0.05). However, during HS, LP tended to decrease the plasma ghrelin concentration compared with NP (P< 0.1). Unlike TN condition, LP had a decreased muscle sirtuin and cytochrome c oxidase transcript than NP during HS (P< 0.05). Our data provide evidence that low protein diets mitigate the negative outcome of heat stress by reducing feed intake and heat production, which are regulated through genes expressed in the gut and skeletal muscle.

Low birth weight (LBW) is associated with reduced postnatal growth efficiency, glucose intolerance and type II diabetes mellitus in adulthood. Using a piglet model of LBW, the objective of this study was to investigate the effect of supplemental Trp on growth, energy balance and blood metabolites in LBW pigs. LBW (< 1.0 Kg) and normal birth weight (NBW; > 1.0 Kg) pigs were selected from twelve sows (parity=2–4, litter size=14–18; three LBW and one NBW piglets/sow) and randomly assigned to 4 milk-replacer based treatments (n=7–8; 7 days old) including: 1) NBW with 0% L-Trp (NBW-T0), 2) LBW with 0% L-Trp (LBW-T0), 3) LBW with 0.4% L-Trp (LBW-T0.4), and 4) LBW with 0.8% L-Trp (LBW-T0.8) for 3 weeks. Diets were provided 4–5 times/day and the voluntary intake was measured for each feeding throughout the study. Growth parameters, heat production and body weight were recorded biweekly. After an overnight fast at week 3, pigs were allowed to consume their respective diets for 1 hour and blood samples were collected at baseline (0), 60 and 120 min after the meal test. The data were analyzed using univariate GLM with Dunnett’s post-hoc test (SPSS®). No significant differences were observed on growth parameters (i.e. body weight, heart girth, wither height and body length), growth performance (i.e. average daily gain, average daily feed intake, gain:feed, gain:protein) and heat production when LBW-T0 was compared with LBW-T0.4 and LBW-T0.8. The plasma triglycerides for pigs fed LBW T0.4 and T0.8 was lower compared to that offered LBW-T0, but total cholesterol did not differ across groups. Pigs in LBW-T0.8 group had lower blood glucose than those fed LBW-T0 at 60 min after the meal challenge. In conclusion, Trp supplementation did not affect the growth and energy balance, but reduced the blood glucose and triglycerides concentration in LBW pigs.

Moderately low protein (MLP) diets can help decrease nutrient excretion from the swine production. However, MLP diets negatively impact growth performance. We hypothesized that supplementing MLP diets with phytogenics may reduce the negative effects of these diets on growth. The objective of this study was to investigate the effect of a phytogenic water additive (PWA; Herbanimal®) on growth performance, blood metabolite and gene expression of amino acids transporters in pigs fed with MLP diets. Forty-eight weaned barrows were allotted to six dietary treatments (n = 8) for 4 weeks: >CON-NS: standard protein diet-no PWA; CON-LS: standard protein diet-low PWA dose (4 ml/L); CON-HS: standard protein diet-high PWA dose (8 ml/L); LP-NS: low protein diet-no PWA; LP-LS: low protein diet-low PWA dose (4 ml/L); LP-HS: low protein diet- high PWA dose (8 ml/L). Feed intake and body weight were recorded daily and weekly, respectively. At week 4, blood and tissue samples were collected and analyzed for metabolites using a chemistry analyzer and amino acid transporters using qPCR, respectively. The data were analyzed by univariate GLM (SPSS®) and the means were separated using paired Student’s t-test corrected by Benjamini-Hochberg. Pigs fed CON-HS improved the average daily gain and serum calcium and phosphorus concentrations compared to CON-NS. Pigs fed LP-LS had higher serum phosphorus and blood urea nitrogen compared to the pigs fed with LP-NS. The mRNA abundance of SLC7A11 in the jejunum was lower in CON-LS and CON-HS compared to CON-NS. Additionally, mRNA abundance of SLC6A19 in the jejunum of pigs fed with LP-LS was higher compared to LP-NS and lower in CON-HS relative to pigs fed with CON-LS. In conclusion, PWA improved the growth performance of pigs fed standard protein diets but not low protein diets. Further, the PWA improved the concentrations of blood calcium and phosphorous in pigs fed MLP diets. Funding: Agrivida and Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals from the USDA-NIFA.