Development and Properties of Francisella tularensis Subsp. holarctica 15 NIIEG Vaccine Strain without the recD Gene

The genomic analysis of all subspecies F. tularensis, as found in Gen Bank NCBI, reveals the presence of genes encoding proteins like to the multifunctional RecBCD enzyme complex in E. coli and other bacteria. To date, the role of the recD gene in F. tularensis, which encodes the alpha chain of exonuclease V, in DNA metabolism processes, has not been studied either in vitro or in vivo. F. tularensis subsp. holarctica 15 NIIEG, a vaccine strain, served as the basis to create the F. tularensis 15D strain with recD deletion. The lack of the recD gene suppresses the integration of suicide plasmids with F. tularensis genome fragments into the chromosome. The modified strain showed reduced growth in vitro and in vivo. This study shows that such deletion significantly reduces the virulence of the strain in BALB/c mice.

EFECTO DE LA SUPLEMENTACIÓN ENZIMÁTICA EN PARÁMETROS PRODUCTIVOS DE CUYES (LÍNEA SINTÉTICA P 0.625)

The aim of this study was to evaluate the effect of the supplementation of an enzymatic complex in integral diets for guinea pigs in the growing phase on productive performance. In total, 54 male guinea pigs (Synthetic line P 0.625) were used and submitted to a diet free of the enzyme complex (T1), and two experimental ones, one with 0.1% (T2) and the other with 0.2% (T3) of enzyme complex evaluated between two weeks of age (weaning) and after rearing (nine weeks old). It was statically evaluated and there were found no significant difference between treatments in relation to final body weight, weight gain, feed intake and feed conversion; and it was reported the weekly average of percentage of dairy dry matter intake by body weight.

Enzyme complex addition in barley or rye broiler diets with two energy levels fed from 1 to 21 days

Two experiments were conducted to evaluate diet digestibility, performance, digestive parameters, and blood parameters when an enzyme complex (EC) was used in barley- and rye-based diets with different energy levels. In the digestibility assay (exp. I), 108 seventeen-day-old Cobb male broilers were distributed in a completely randomized design in 2 × 2 × 2 + 1 factorial arrangement with two feeds (barley or rye), two EC levels (0% and 0.02%), and two energy levels [3025 and 3125 kcal apparent metabolizable energy (AME)·kg−1], plus a control treatment. In exp. II, 1080 one-day-old Cobb male broilers were distributed in a completely randomized design in 2 × 2 × 2 + 1 factorial arrangement with two feeds (barley or rye), two EC levels (0% and 0.02%), and two energy levels (2875 and 2975 kcal AME·kg−1). No interactions were observed for any variables (exp. I and II). Enzyme complex improved the apparent metabolizable coefficient of gross energy (P = 0.0432) of diets. The EC provided greater weight gain (P = 0.0003) and better feed conversion (P = 0.0025). Intestinal viscosity at 21 d was reduced (P < 0.0001) with the addition of the EC. The EC improved nutrient digestibility and performance, but the effects of energy reduction on performance could not be overcome.

The low-complexity domains of the KMT2D protein regulate histone monomethylation transcription to facilitate pancreatic cancer progression

Background Liquid–liquid phase separation (LLPS) within the nucleus is directly linked to driving gene expression through transcriptional complexes. Histone lysine methyltransferase 2D (KMT2D) is widely present in many cancers. It is known to epigenetically stimulate the expression of genes associated with tumorigenesis and metastasis. Our analyses show that KMT2D possesses two distinct low-complexity domains (LCDs) capable of driving the assembly of membrane-less condensates. The dependence of the mechanisms underlying monomethylation of H3K4 on the LLPS microenvironment derived from KMT2D LCDs is unclear in tumor. Methods KMT2D LCD-depletion cells were used to investigate tumor cell proliferation, apoptosis, and migration. We identified some core proteins, including WDR5, RBBP5, and ASH2L, which are involved in the KMT2D-associated catalytic complex in KMT2D LCD-deficient cells to further elucidate the mechanism that decreases monomethylation of H3K4. We also evaluated the viability of KMT2D LCD-deficient cells in vivo. Finally, using 1,6-hexanediol (HD), an inhibitor of LLPS, we determined cell activities associated with KMT2D function in wild-type PANC-1 cells. Results Without the LLPS microenvironment in KMT2D LCD-deficient cells or wild-type PANC-1 cells treated with HD, the WDR5 protein was significantly less stable and the protein–protein interactions between the components of the KMT2D–enzyme complex were attenuated, impairing the formation of the complex. Moreover, with the decrease in H3K4me1 level at enhancers, transcription factors such as LIFR and KLF4 were markedly downregulated, effectively inhibiting tumor progression. In xenograft tumor models, PANC-1 cells lacking the KMT2D LCDs showed effectively suppressed tumor growth compared to normal cells. Conclusions Our data indicate that the two low-complexity domains of the KMT2D protein could form a stable LLPS microenvironment, promoting the KMT2D catalysis of H3K4 monomethylation through stabilization of the WDR5 protein and KMT2D–enzyme complex. Therefore, finding ways to regulate the LLPS microenvironment will be benefitial for new cancer treatment strategies.

1286. Taniborbactam Inhibits Cefepime-Hydrolyzing Variants of Pseudomonas-derived Cephalosporinase (PDC)

Background PDC is a class C β-lactamase in P. aeruginosa. PDC-88 is a variant characterized by a Thr-Pro amino acid deletion in the R2-loop (Δ289-290; Fig. 1). This deletion reduces susceptibility to cefepime (FEP), ceftazidime (CAZ), and ceftolozane-tazobactam (TOL/TZB), but the mechanism for this “gain of function” is unknown. Taniborbactam (TAN) is a novel cyclic boronate β-lactamase inhibitor (BLI) with activity against all four β-lactamase classes and is currently undergoing a phase 3 clinical trial paired with FEP. Herein, we studied the extended-spectrum AmpC (ESAC) phenotype of PDC-88 and examined the ability of TAN to inhibit this variant. Structure of PDC-1 (PDB ID: 4GZB) with PDC-88 deleted residues in red and substitutions in green. All four amino acid substitutions (T79A, V178L, V329I, and G346A) are common (occurring in 10% or more of PDC variants) and have not been associated with resistance. Image rendered using UCSF Chimera. Methods Broth microdilution minimum inhibitory concentrations (MIC) were determined in accordance with CLSI. PDC-3 and PDC-88 were purified, and steady-state enzyme kinetics were determined. Quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was performed. Results In isogenic E. coli expressing PDC-3 or PDC-88, FEP MIC increased 8- or 128-fold, respectively, compared to the empty vector. Addition of TAN at 4 μg/ml restored FEP activity with MIC lowered to 0.25 μg/ml (Table 1) for both PDC-3 and PDC-88 bearing strains. PDC-88 demonstrated a 9-fold lower KM, 3.4-fold lower kcat, and 2.6-fold higher kcat/KM for FEP compared to PDC-3 (Table 2A). TAN Ki values were 4- to 10-fold lower than avibactam (AVI) and 40- to 95-fold lower than TZB. The TAN acylation constant (k2/K) was 7- to 12-fold greater than AVI and 133- to 366-fold higher than TZB (Table 2B). Q-TOF-MS revealed faster deacylation of FEP by PDC-88 compared to TOL and CAZ. TOL was acylated and deacylated by PDC-88 but not by PDC-3. CAZ was readily acylated but slowly deacylated by PDC-88 compared to PDC-3 (Fig. 2). Cefepime Minimum Inhibitory Concentration (MIC) for PDC-1 and a series of partial R2-loop deletions with and without taniborbactam, avibactam, and tazobactam. In all variants, taniborbactam and avibactam restored susceptiblity while tazobactam is less effective against PDC-88 and variants. Summary of kinetic constants. (A) Comparison of Michaelis constant (KM), turnover number (kcat), and catalytic efficiency (kcat/KM) of nitrocefin and cefepime with PDC-3 and PDC-88. (B) Comparison of inhibition constant (Ki) and acylation constant (k2/K) for avibactam, tazobactam, and taniborbactam with PDC-3 and PDC-88. Graphical summary of mass spectrometry results for substrate acyl-enzyme complex capture experiments. FEP, cefepime; CAZ, ceftazidime; TOL, ceftolozane. Primes indicate a modified substrate (loss of R2 group). TOL does not form an acyl-enzyme complex with PDC-3. Conclusion Different kinetic constants are responsible for the elevated cephalosporin MICs. We posit that PDC-88 increases FEP MIC by enhanced hydrolysis; TOL MICs by enabling acylation; and CAZ MICs by both trapping and enhanced hydrolysis. TAN inhibits both PDC-3 and PDC-88 with similar kinetic profiles. Notably, TAN appears to be a more efficient inhibitor of PDC than current BLIs targeted for use against P. aeruginosa (lower Ki, higher k2/K values). The combination of TAN and FEP may represent an important treatment option for P. aeruginosa isolates that develop ESAC phenotypes. Disclosures Focco van den Akker, PhD, Venatorx Pharmaceuticals, Inc. (Grant/Research Support) Brittany A. Miller, BS, Venatorx Pharmaceuticals, Inc. (Employee) Tsuyoshi Uehara, PhD, Venatorx Pharmaceuticals, Inc. (Employee) David A. Six, PhD, Venatorx Pharmaceuticals, Inc. (Employee) Krisztina M. Papp-Wallace, Ph.D., Merck & Co., Inc. (Grant/Research Support)Spero Therapeutics, Inc. (Grant/Research Support)Venatorx Pharmaceuticals, Inc. (Grant/Research Support)Wockhardt Ltd. (Other Financial or Material Support, Research Collaborator) Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.