Induction of the antioxidant defense system using long-chain carotenoids extracted from extreme halophilic archaeon, Halovenus aranensis.

The field of microbial pigments is an emerging area in natural products science. Carotenoids form a major class of such pigments and are found to be diversely synthesized by microorganisms that reside in hypersaline ecosystems to provide resistance against oxidative stress. Human cells can benefit from compounds such as carotenoids as antioxidant agents through either their capability to quench free radicals or their effect on promoting the antioxidant defense pathway. In this study, the antioxidant effectiveness of carotenoid extract from an extremely halophilic archaeon Halovenus aranensis strain EB27T has been evaluated using different approaches. Finally, the ability of the extracted pigment to induce the antioxidant defense pathway of human primary skin fibroblast cells was studied. Hvn. aranensis carotenoid extract exhibited strong effectiveness such that at 2 µg/ml, the carotenoid extract fully neutralized the oxidative stress of hydrogen peroxide at its EC50 based on MTT assay. Results from real-time PCR of relevant genes, luciferase bioreporter of oxidative stress, and the western blot analysis further confirmed the antioxidant capability of the carotenoids. It was also shown the carotenoid extract had more antioxidant activity compared to ß-carotene the same concentration. Results suggest the carotenoid extract from this archaeon to have high potential for clinical and industrial applications.

A Mildly Acidic Environment Alters Pseudomonas aeruginosa Virulence and Causes Remodeling of the Bacterial Surface.

Pseudomonas aeruginosa is a versatile pathogen that resists environmental stress, such as suboptimal pH. As a result of exposure to environmental stress, P. aeruginosa shows an altered virulence-related phenotype. This study investigated the modifications that P. aeruginosa undertakes at a mildly low pH (pH 5.0) compared with the bacteria grown in a neutral medium (pH 7.2). Results indicated that in a mildly acidic environment, expression of two-component system genes (phoP/phoQ and pmrA/pmrB), lipid A remodeling genes such as arnT and pagP and virulence genes, i.e., pqsE and rhlA, were induced. Moreover, lipid A of the bacteria grown at a mildly low pH is modified by adding 4-amino-arabinose (l-Ara4N). Additionally, the production of virulence factors such as rhamnolipid, alginate, and membrane vesicles is significantly higher in a mildly low-pH environment than in a neutral medium. Interestingly, at a mildly low pH, P. aeruginosa produces a thicker biofilm with higher biofilm biomass. Furthermore, studies on inner membrane viscosity and permeability showed that a mildly low pH causes a decrease in the inner membrane permeability and increases its viscosity. Besides, despite the importance of PhoP, PhoQ, PmrA, and PmrB in Gram-negative bacteria for responding to low pH stress, we observed that the absence of each of these two-component systems does not meaningfully impact the remodeling of the P. aeruginosa envelope. Given that P. aeruginosa is likely to encounter mildly acidic environments during infection in its host, the alterations that the bacterium undertakes under such conditions must be considered in designing antibacterial strategies against P. aeruginosa. IMPORTANCE P. aeruginosa encounters environments with acidic pH when establishing infections in hosts. The bacterium develops an altered phenotype to tolerate a moderate decrease in the environmental pH. At the level of the bacterial envelope, modified lipid A composition and a reduction of the bacterial inner membrane permeability and fluidity are among the changes P. aeruginosa undergoes at a mildly low pH. Also, the bacterium is more likely to form biofilm in a mildly acidic environment. Overall, these alterations in the P. aeruginosa phenotype put obstacles in the way of antibacterial activities. Thus, considering physiological changes in the bacterium at low pH helps design and implement antimicrobial approaches against this hostile microorganism.

Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains.

Transfusion of red blood cell concentrates is the most common medical procedure to treat anaemia. However, their storage is associated with development of storage lesions, including the release of extracellular vesicles. These vesicles affect in vivo viability and functionality of transfused red blood cells and appear responsible for adverse post-transfusional complications. However, the biogenesis and release mechanisms are not fully understood. We here addressed this issue by comparing the kinetics and extents of extracellular vesicle release as well as red blood cell metabolic, oxidative and membrane alterations upon storage in 38 concentrates. We showed that extracellular vesicle abundance increased exponentially during storage. The 38 concentrates contained on average 7 × 1012 extracellular vesicles at 6 weeks (w) but displayed a ∼40-fold variability. These concentrates were subsequently classified into 3 cohorts based on their vesiculation rate. The variability in extracellular vesicle release was not associated with a differential red blood cell ATP content or with increased oxidative stress (in the form of reactive oxygen species, methaemoglobin and band3 integrity) but rather with red blood cell membrane modifications, i.e., cytoskeleton membrane occupancy, lateral heterogeneity in lipid domains and transversal asymmetry. Indeed, no changes were noticed in the low vesiculation group until 6w while the medium and the high vesiculation groups exhibited a decrease in spectrin membrane occupancy between 3 and 6w and an increase of sphingomyelin-enriched domain abundance from 5w and of phosphatidylserine surface exposure from 8w. Moreover, each vesiculation group showed a decrease of cholesterol-enriched domains associated with a cholesterol content increase in extracellular vesicles but at different storage time points. This observation suggested that cholesterol-enriched domains could represent a starting point for vesiculation. Altogether, our data reveal for the first time that the differential extent of extracellular vesicle release in red blood cell concentrates did not simply result from preparation method, storage conditions or technical issues but was linked to membrane alterations.

Corrigendum: Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains.

[This corrects the article DOI: 10.3389/fphys.2023.1205493.].

Contribution of Membrane Vesicle to Reprogramming of Bacterial Membrane Fluidity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen capable of resisting environmental insults by applying various strategies, including regulating membrane fluidity and producing membrane vesicles (MVs). This study examined the difference in membrane fluidity between planktonic and biofilm modes of growth in P. aeruginosa and whether the ability to alter membrane rigidity in P. aeruginosa could be transferred via MVs. To this end, planktonic and biofilm P. aeruginosa were compared with respect to the lipid composition of their membranes and their MVs and the expression of genes contributing to alteration of membrane fluidity. Additionally, viscosity maps of the bacterial membrane in planktonic and biofilm lifestyles and under the effect of incubation with bacterial MVs were obtained. Further, the growth rate and biofilm formation capability of P. aeruginosa in the presence of MVs were compared. Results showed that the membrane of the biofilm bacteria is significantly less fluid than the membrane of the planktonic bacteria and is enriched with saturated fatty acids. Moreover, the enzymes involved in altering the structure of existing lipids and favoring membrane rigidification are overexpressed in the biofilm bacteria. MVs of biofilm P. aeruginosa elicit membrane rigidification and delay the bacterial growth in the planktonic lifestyle; conversely, they enhance biofilm development in P. aeruginosa. Overall, the study describes the interplay between the planktonic and biofilm bacteria by shedding light on the role of MVs in altering membrane fluidity. IMPORTANCE Membrane rigidification is a survival strategy in Pseudomonas aeruginosa exposed to stress. Despite various studies dedicated to the mechanism behind this phenomenon, not much attention has been paid to the contribution of the bacterial membrane vesicles (MVs) in this regard. This study revealed that P. aeruginosa rigidifies its membrane in the biofilm mode of growth. Additionally, the capability of decreasing membrane fluidity is transferable to the bacterial population via the bacterial MVs, resulting in reprogramming of bacterial membrane fluidity. Given the importance of membrane rigidification for decreasing the pathogen's susceptibility to antimicrobials, elucidation of the conditions leading to such biophysicochemical modulation of the P. aeruginosa membrane should be considered for the purpose of developing therapeutic approaches against this resistant pathogen.

The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence.

Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716.

Membrane Vesicle Production as a Bacterial Defense Against Stress.

Membrane vesicles are the nano-sized vesicles originating from membranes. The production of membrane vesicles is a common feature among bacteria. Depending on the bacterial growth phase and environmental conditions, membrane vesicles show diverse characteristics. Various physiological and ecological roles have been attributed to membrane vesicles under both homeostatic and stressful conditions. Pathogens encounter several stressors during colonization in the hostile environment of host tissues. Nutrient deficiency, the presence of antibiotics as well as elements of the host's immune system are examples of stressors threatening pathogens inside their host. To combat stressors and survive, pathogens have established various defensive mechanisms, one of them is production of membrane vesicles. Pathogens produce membrane vesicles to alleviate the destructive effects of antibiotics or other types of antibacterial treatments. Additionally, membrane vesicles can also provide benefits for the wider bacterial community during infections, through the transfer of resistance or virulence factors. Hence, given that membrane vesicle production may affect the activities of antibacterial agents, their production should be considered when administering antibacterial treatments. Besides, regarding that membrane vesicles play vital roles in bacteria, disrupting their production may suggest an alternative strategy for battling against pathogens. Here, we aim to review the stressors encountered by pathogens and shed light on the roles of membrane vesicles in increasing pathogen adaptabilities in the presence of stress-inducing factors.

Author Correction: Designing a whole cell bioreporter to show antioxidant activities of agents that work by promotion of the KEAP1-NRF2 signaling pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Designing a whole cell bioreporter to show antioxidant activities of agents that work by promotion of the KEAP1-NRF2 signaling pathway.

The major signaling pathway in human cells is related to the antioxidant defense system. The main component of this system is a transcription factor, Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). It regulates this system in different cellular situations under stimulation by oxidative stress or antioxidants. Thus, detecting the stimulation of NRF2 via a screening strategy may enable us to discover stimulating agents of NRF2-related signaling pathway. With this in mind, we designed a whole cell bioreporter containing the NRF2 response elements that are inserted in a luciferase vector, immediately upstream of a luciferase gene whose promoter has been removed. This bioreporter is activated by stimulators such as 3H-1,2-dithiole-3-thione (D3T), butyl hydroxyanisole (BHA) and ascorbic acid reacting as antioxidant agents. It was observed that the regulatory region of the NRF2 gene, which is identified by NRF2 protein, is located inside its coding region. This designed bioreporter can detect the presence of antioxidant agents. It also exhibits a significant linear correlation over different doses of these agents ranging from 0.8 to 80 µM for ascorbic acid, 0.1 to 100 µM for D3T, and 0.1 to 100 µM for BHA. This detection system is proven to be more sensitive than Real-time PCR, suggesting it to be a highly sensitive system among the available methods.