Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. cremoris during milk fermentation

Background: Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance. This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. cremoris MG1363 at the transcriptional and metabolic levels in relation to acidification kinetics and growth conditions, especially at an early stage of growth. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. Results: Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L. lactis MG1363 strain. Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. lactis MG1363 strain was supported by a coherent and early adaptation to oxygen after 1 hour of culture at both gene expression and metabolic levels. In oxygen metabolism, the over-expression of all the genes of the nrd (ribonucleotide reductases) operon or fhu (ferrichrome ABC transports) genes was particularly significant. In carbon metabolism, the presence of oxygen led to an early shift at the gene level in the pyruvate pathway towards the acetate/2,3-butanediol pathway confirmed by the kinetics of metabolite production. Finally, the MG1363 strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage. Conclusions: Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. lactis to initial oxidative stress. An early and transitional adaptation to oxidative stress was revealed for L. lactis subsp. cremoris MG1363 in the presence of initially high levels of oxygen. This enables the cells to maintain key traits that are of great importance for industry, such as rapid acidification and reduction of the redox potential of the growth media

Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix

International audienceIn complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS

The circadian clock, metabolism and obesity

In the last decades, obesity has been on the rise becoming a burden for health care systems. The reasons behind this rise are most likely caused by lifestyle rather than by an increase in gene mutations, because manifestations of genetic alterations would take longer than just a few decades. Lifestyle has a great impact on the circadian system and therefore on the body internal organization of physiological and biochemical processes, regulating various aspects of behavior and metabolism. In the following, I will discuss recent studies delineating relationships between metabolic processes and the circadian system, how metabolites and nutrients regulate the circadian clock and how nuclear receptors can act as metabolic sensors and clock regulators. Finally, I will discuss how clock modulation and feeding patterns influence the development of obesity

A Temporal -omic Study of Propionibacterium freudenreichii CIRM-BIA1T Adaptation Strategies in Conditions Mimicking Cheese Ripening in the Cold

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It grows when cheeses are ripened in a warm room (about 24°C). Cheeses with an acceptable eye formation level are transferred to a cold room (about 4°C), inducing a marked slowdown of propionic fermentation, but P. freudenreichii remains active in the cold. To investigate the P. freudenreichii strategies of adaptation and survival in the cold, we performed the first global gene expression profile for this species. The time-course transcriptomic response of P. freudenreichii CIRM-BIA1T strain was analyzed at five times of incubation, during growth at 30°C then for 9 days at 4°C, under conditions preventing nutrient starvation. Gene expression was also confirmed by RT-qPCR for 28 genes. In addition, proteomic experiments were carried out and the main metabolites were quantified. Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from 30°C to 4°C (P<0.05 and |fold change|>1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1T strain over-expressed genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis, and in glycogen synthesis. Interestingly, the expression of different genes involved in the formation of important cheese flavor compounds, remained unchanged at 4°C. This could explain the contribution of P. freudenreichii to cheese ripening even in the cold. In conclusion, P. freudenreichii remains metabolically active at 4°C and induces pathways to maintain its long-term survival

Regulation of basal cellular physiology by the homeostatic unfolded protein response

The extensive membrane network of the endoplasmic reticulum (ER) is physically juxtaposed to and functionally entwined with essentially all other cellular compartments. Therefore, the ER must sense diverse and constantly changing physiological inputs so it can adjust its numerous functions to maintain cellular homeostasis. A growing body of new work suggests that the unfolded protein response (UPR), traditionally charged with signaling protein misfolding stress from the ER, has been co-opted for the maintenance of basal cellular homeostasis. Thus, the UPR can be activated, and its output modulated, by signals far outside the realm of protein misfolding. These findings are revealing that the UPR causally contributes to disease not just by its role in protein folding but also through its broad influence on cellular physiology

Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don't Forget the Fuel.

T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors

The Therapeutic Potential of T Cell Metabolism

Transplant rejection mediated by the adaptive immune system remains a major barrier to achieving long-term tolerance and graft survival. Emerging evidence indicates that lymphocytes rapidly shift their metabolic programs in response to activation, co-stimulatory, and cytokine signals to support required effector cell differentiation and function. These observations have led to the hypothesis that manipulating the metabolic programs of immune cells could serve as a powerful therapeutic strategy for attenuating deleterious immune responses and facilitating durable tolerance in the setting of allogeneic solid organ or bone marrow transplant. In this mini-review, we introduce the fundamentals of metabolism, highlight the current understanding of how adaptive immune cells utilize their metabolic programs, and discuss the potential for targeting metabolism as a therapeutic approach to induce tolerance in the transplant setting

Metabolic Adaptation of Human CD4+ and CD8+ T-Cells to T-Cell Receptor-Mediated Stimulation

Linking immunometabolic adaptation to T-cell function provides insight for the development of new therapeutic approaches in multiple disease settings. T-cell activation and downstream effector functions of CD4+ and CD8+ T-cells are controlled by the strength of interaction between the T-cell receptor (TCR) and peptides presented by human leukocyte antigens (pHLA). The role of TCR-pHLA interactions in modulating T-cell metabolism is unknown. Here for the first time we explore the relative contributions of the main metabolic pathways to functional responses in human CD4+ and CD8+ T-cells. Increased expression of hexokinase II accompanied by higher basal glycolysis is demonstrated in CD4+ T-cells; cytokine production in CD8+ T-cells is more reliant on oxidative phosphorylation. Using antigen-specific CD4+ and CD8+ T cell clones and altered peptide ligands we demonstrate that binding affinity tunes the underlying metabolic shift. Overall this study provides important new insight into how metabolic pathways are controlled during antigen-specific activation of human T-cells