The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Human Tendon Stem/Progenitor Cell Features and Functionality Are Highly Influenced by in vitro Culture Conditions

Our understanding of tendon biology continues to evolve, thus leading to opportunities for developing novel, evidence-based effective therapies for the treatment of tendon disorders. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. We described different molecular and phenotypic profiles of TSPCs modulated by culture density, as well as their multipotency and secretory activities. Moreover, in the same experimental setting, we evaluated for different responses to inflammatory stimuli mediated by TNFα and IFNγ. We also preliminarily investigated their immunomodulatory activity and their role in regulating degradation of substance P. Our findings indicated that TSPCs cultured at low density (LD) exhibited cobblestone morphology and a reduced propensity to differentiate. A distinctive immunophenotypic profile was also observed with high secretory and promising immunomodulatory responses when primed with TNFα and IFNγ. In contrast, TSPCs cultured at high density (HD) showed a more elongated fibroblast-like morphology, a greater adipogenic differentiation potential, and a higher expression of tendon-related genes with respect to LD. Finally, HD TSPCs showed immunomodulatory potential when primed with TNFα and IFNγ, which was slightly lower than that shown by LD. A shift from low to high culture density during TSPC expansion demonstrated intermediate features confirming the cellular adaptability of TSPCs. Taken together, these experiments allowed us to identify relevant differences in TSPCs based on culture conditions. This ability of TSPCs to acquire distinguished morphology, phenotype, gene expression profile, and functional response advances our current understanding of tendons at a cellular level and suggests responsivity to cues in their in situ microenvironment.

The influence of density on survival and larval development in the sea urchin Arbacia dufresnii (Echinodermata: Echinoidea)

Introduction: Density is one of the critical factors in echinoderm larvae for aquaculture purposes. Echinoplutei larvae are very sensitive to overcrowding. High culture density can lead to problems with bacteria or protozoa, decreasing survival and generating abnormal morphotypes. Objective: To evaluate the effect of culture density on survival and larval growth in the sea urchin Arbacia dufresnii. Methods: Two days after fertilization of A. dufresnii we we kept treatments at 1, 3, 5 and 10 larvae.ml-1, with three replicates each. We recorded survival and abnormal morphotypes periodically, as well as growth:somatic rod length, total length, and length of the post oral arms,. we applied generalized linear models. Results: Survival is dependent on density, time and replicates, and their interactions. Larval growth depended on density and time, also with interaction between the variables. The treatment of 5 larvae.ml-1 had the highest survival and larval condition. Conclusions: Larval culture of A. dufresnii had the best results at 5 larvae.ml-1.

Effects of mouse fetal liver cell culture density on hematopoietic cell expansion in three-dimensional cocultures with stromal cells

Objective: An effective ex vivo expansion system of primitive hematopoietic cells (HCs) is required for wider application of hematopoietic stem cell transplantation. In this study, we examined effects of culture density on mouse fetal liver cells (FLCs) used as an HC source for the expansion of primitive HCs in three-dimensional (3D) cocultures with two kinds of mouse stromal cell lines (OP9 or C3H10T1/2). Materials and methods: FLCs were seeded at different densities (1, 2, and 10 × 107 cells/cm3) into porous polymer scaffolds with or without stromal cell layers and HCs were expanded in the cultures for 2 weeks without exogenous cytokines. Results: Differential effects of culture density on HC expansion were observed between cocultures and solitary FLC controls. In stromal cell cocultures, high expansion of HCs was achieved when FLCs were seeded at low densities. In contrast, the expansion in the controls was enhanced with increasing culture densities. With respect to expansion of primitive HCs existing in the FLCs, cocultures with C3H10T1/2 cells were superior to those with OP9 cells with a 29.3-fold expansion for c-kit+ hematopoietic progenitor cells and 8.3-fold expansion for CD34+ hematopoietic stem cells. In the controls, HC expansion was lower than in any cocultures, demonstrating the advantages of coculturing for HC expansion. Conclusion: Stromal cell lines are useful in expanding primitive HCs derived from FLCs in 3D cocultures. Culture density is a pivotal factor for the effective expansion of primitive HCs and this effect differs by culture condition.

Environmental Regulation of PndbA600, an Auto-Inducible Promoter for Two-Stage Industrial Biotechnology in Cyanobacteria

Cyanobacteria are photosynthetic prokaryotes being developed as sustainable platforms that use renewable resources (light, water, and air) for diverse applications in energy, food, environment, and medicine. Despite the attractive promise that cyanobacteria offer to industrial biotechnology, slow growth rates pose a major challenge in processes which typically require large amounts of biomass and are often toxic to the cells. Two-stage cultivation strategies are an attractive solution to prevent any undesired growth inhibition by de-coupling biomass accumulation (stage I) and the industrial process (stage II). In cyanobacteria, two-stage strategies involve costly transfer methods between stages I and II, and little work has been focussed on using the distinct growth and stationary phases of batch cultures to autoregulate stage transition. In the present study, we identified and characterised a growth phase-specific promoter, which can serve as an auto-inducible switch to regulate two-stage bioprocesses in cyanobacteria. First, growth phase-specific genes were identified from a new RNAseq dataset comparing two growth phases and six nutrient conditions in Synechocystis sp. PCC 6803, including two new transcriptomes for low Mg and low K. A type II NADH dehydrogenase (ndbA) showed robust induction when the cultures transitioned from exponential to stationary phase growth. Behaviour of a 600-bp promoter sequence (PndbA600) was then characterised in detail following the expression of PndbA600:GFP in Synechococcus sp. PCC 7002. Culture density and growth media analyses showed that PndbA600 activation was not dependent on increases in culture density per se but on N availability and on another activating factor present in the spent media of stationary phase cultures (Factor X). PndbA600 deactivation was dependent on the changes in culture density and in either N availability or Factor X. Electron transport inhibition studies revealed a photosynthesis-specific enhancement of active PndbA600 levels. Our findings are summarised in a model describing the environmental regulation of PndbA600, which can now inform the rational design of two-stage industrial processes in cyanobacteria.