HHP-treated Cells Research Articles

Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis.

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+ Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells.IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

Listeria monocytogenes cells injured by high hydrostatic pressure and their recovery in nutrient-rich or -free medium during cold storage

ABSTRACTCells of Listeria monocytogenes suspended in phosphate-buffered saline (PBS) were treated by high hydrostatic pressure (HHP; 500 MPa, 25°C, 10 min), diluted by ten folds using trypticase soy broth (TSB) or PBS, and stored at cold temperatures of 0–15°C. Viable cell count in TSB increased logarithmically close to the initial count at each storage temperature, while that in PBS increased temporarily and subsequently decreased to almost nondetectable level except the case at 15°C, where it showed logarithmic increase thereafter. Based on proliferation experiments where their healthy cells were inoculated to TSB or to PBS containing their heat-killed dead cells, it was suggested that increase in the viable count of HHP-treated cells in TSB and PBS could be ascribed to the recovery of colony forming ability and/or proliferation depending on the cold storage temperature.

Metabolome analysis of Escherichia coli ATCC25922 cells treated with high hydrostatic pressure at 400 and 600 MPa

Escherichia coli cells were treated with high hydrostatic pressure (HHP) at 400 and 600MPa. Metabolites (70-1027m/z) extracted from HHP-treated cells were analyzed using capillary electrophoresis-time-of-flight mass spectrometry and were compared with those extracted from control cells (not treated with HHP). A total of 133 metabolites were identified and mapped to metabolic pathways, and many of these (42.1%) decreased due to the HHP treatment, including NAD+, NADP+, ATP, and substrates for DNA synthesis. Principal component analysis suggested that the central sugar and nucleic acid metabolic pathways were strongly influenced by HHP. A bottleneck in the central sugar metabolic pathway was observed in HHP-treated cells, which created a metabolic imbalance; metabolites mapped upstream (glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate) were accumulated and those downstream (3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate) were depleted. Ribonucleotides were decreased, but the reduction was moderate compared with that of substrates for DNA synthesis; the exception was ATP, which also substantially decreased. The bottleneck in the glycolytic pathway partly explained the exhaustion of ATP. NAD+/NADH ratio of HHP treated cells was comparable with that of untreated control cells.

Characterization of high hydrostatic pressure-injured Bacillus subtilis cells.

High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250MPa of HHP for 10min at 25°C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations≥100mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.

Dendritic cells pulsed with tumor cells killed by high hydrostatic pressure induce strong immune responses and display therapeutic effects both in murine TC-1 and TRAMP-C2 tumors when combined with docetaxel chemotherapy.

High hydrostatic pressure (HHP) has been shown to induce immunogenic cell death of cancer cells, facilitating their uptake by dendritic cells (DC) and subsequent presentation of tumor antigens. In the present study, we demonstrated immunogenicity of the HHP-treated tumor cells in mice. HHP was able to induce immunogenic cell death of both TC-1 and TRAMP-C2 tumor cells, representing murine models for human papilloma virus-associated tumors and prostate cancer, respectively. HHP-treated cells induced stronger immune responses in mice immunized with these tumor cells, documented by higher spleen cell cytotoxicity and increased IFNγ production as compared to irradiated tumor cells, accompanied by suppression of tumor growth in vivo in the case of TC-1 tumors, but not TRAMP-C2 tumors. Furthermore, HHP-treated cells were used for DC-based vaccine antigen pulsing. DC co-cultured with HHP-treated tumor cells and matured by a TLR 9 agonist exhibited higher cell surface expression of maturation markers and production of IL-12 and other cytokines, as compared to the DC pulsed with irradiated tumor cells. Immunization with DC cell-based vaccines pulsed with HHP-treated tumor cells induced high immune responses, detected by increased spleen cell cytotoxicity and elevated IFNγ production. The DC-based vaccine pulsed with HHP-treated tumor cells combined with docetaxel chemotherapy significantly inhibited growth of both TC-1 and TRAMP-C2 tumors. Our results indicate that DC-based vaccines pulsed with HHP-inactivated tumor cells can be a suitable tool for chemoimmunotherapy, particularly with regard to the findings that poorly immunogenic TRAMP-C2 tumors were susceptible to this treatment modality.

Synergistic Effect of High Hydrostatic Pressure and Natural Antimicrobials on Inactivation Kinetics ofBacillus cereusin a Liquid Whole Egg and Skim Milk Mixed Beverage

An in-depth study was conducted in order to extend the storage life of a liquid whole egg-skim milk (LWE-SM) mixed beverage to enhance its safety and the safety of related beverages. Bacillus cereus vegetative cells (1 x 10(8) colony-forming units [CFU]/mL) were inoculated in LWE-SM beverages with or without natural antimicrobial supplements: flavonol rich-cocoa powder (cocoanOX 12%, CCX) (700 ppm), vanillin (700 ppm), anise (700 ppm), and cinnamon (700 ppm). B. cereus cells were maintained at 10 degrees C for 10 days in the different beverages to test the bacteriostatic or inhibitory effect of the aforementioned ingredients. Beverages were treated with high hydrostatic pressure (HHP) technology and stored at 10 degrees C for 15 days after treatment. All natural antimicrobials reduced the micro(max) values and increased the lag phase time of B. cereus, and Gompertz growth curves showed different inhibitory effects depending on the substance. The maximum inhibitory effect (1.330 log cycle reduction) was achieved in LWE-SM-cinnamon-supplemented beverage. The maximum inactivation achieved by HHP in LWE-SM beverage was a reduction of around 3.89 +/- 0.25 log cycles at 300 MPa for 12 minutes. When supplemented beverages were treated under the same conditions, enhanced inactivation levels were achieved. This increased inactivation can be attributed to a synergistic effect when the LWE-SM was supplemented with flavonol-rich cocoa powder, cinnamon, and vanillin. The maximum synergistic effect was observed in LWE-SM-CCX-supplemented beverage. During the refrigerated storage of B. cereus HHP-treated cells in beverages to which antimicrobials had been added, the inhibitory effect was dependent on the previously applied pressure level.