Physiological Functions of Thiol Peroxidases (Gpx1 and Prdx2) during Xenopus laevis Embryonic Development

Glutathione peroxidase 1 (Gpx1) and peroxiredoxin 2 (Prdx2) belong to the thiol peroxidase family of antioxidants, and have been studied for their antioxidant functions and roles in cancers. However, the physiological significance of Gpx1 and Prdx2 during vertebrate embryogenesis are lacking. Currently, we investigated the functional roles of Gpx1 and Prdx2 during vertebrate embryogenesis using Xenopus laevis as a vertebrate model. Our investigations revealed the zygotic nature of gpx1 having its localization in the eye region of developing embryos, whereas prdx2 exhibited a maternal nature and were localized in embryonic ventral blood islands. Furthermore, the gpx1-morphants exhibited malformed eyes with incompletely detached lenses. However, the depletion of prdx2 has not established its involvement with embryogenesis. A molecular analysis of gpx1-depleted embryos revealed the perturbed expression of a cryba1-lens-specific marker and also exhibited reactive oxygen species (ROS) accumulation in the eye regions of gpx1-morphants. Additionally, transcriptomics analysis of gpx1-knockout embryos demonstrated the involvement of Wnt, cadherin, and integrin signaling pathways in the development of malformed eyes. Conclusively, our findings indicate the association of gpx1 with a complex network of embryonic developmental pathways and ROS responses, but detailed investigation is a prerequisite in order to pinpoint the mechanistic details of these interactions.

Nontypeable Haemophilus influenzae redox recycling of protein thiols promotes resistance to oxidative killing and bacterial survival in biofilms in a smoke related infection model

Smoke exposure is a risk factor for community acquired pneumonia, which is typically caused by host adapted opportunists like nontypeable Haemophilus influenzae (NTHi). Genomic analyses of NTHi revealed homologs of enzymes involved in thiol metabolism, which can have key roles in oxidant resistance. Using a clinical NTHi isolate (NTHi 7P49H1), we generated isogenic mutant bacterial strains in which homologs of glutathione reductase (NTHI 0251), thiol peroxidase (NTHI 0361), thiol peroxidase (NTHI 0907), thioredoxin reductase (NTHI 1327) and glutaredoxin/peroxiredoxin (NTHI 0705) were inactivated. Bacterial protein analyses revealed significant increases in protein oxidation after oxidative stress for all the mutant strains. Similarly, each of these mutants were less resistant to oxidative killing compared with the parental strain; these phenotypes were reversed by genetic complementation. Quantitative confocal analysis of biofilms showed reducted biofilm thickness and density, and significant sensitization of bacteria within the biofilm structure to oxidative killing for thiol mutant strains. Smoke-exposed mice infected with NTHi 7P49H1 showed significantly increased lung bacterial load, as compared to control mice. Immunofluorescent staining of lung tissues showed NTHi communities on the lung mucosa, interspersed with host neutrophil extracellular traps; these bacteria had surface moieties associated with the Hi biofilm matrix, and transcript profiles consistent with NTHi biofilms. In contrast, infection with the panel of NTHi mutants showed significant decrease in lung bacterial load. Comparable results were observed in bactericidal assays with neutrophil extracellular traps in vitro. Thus, we conclude that thiol mediated redox homeostasis promotes persistence of NTHi within biofilm communities.ImportanceChronic bacterial respiratory infections are a significant problem for smoke exposed individuals, especially those with chronic obstructive pulmonary disease (COPD). These infections often persist despite antibiotic use. Thus, the bacteria remain and contribute to the development of inflammation and other respiratory problems. Respiratory bacteria often form biofilms within the lungs, while growing in a biofilm their antibiotic and oxidative stress resistance is incredibly heightened. It is well documented that redox homeostasis genes are upregulated during this phase of growth. Many common respiratory pathogens such as NTHi and Streptococcus pneumoniae are reliant on scavenging from the host the necessary components they need to maintain these redox systems. This work here begins to lay down the foundation for exploiting this requirement and thiol redox homeostasis pathways of these bacteria as a therapeutic target for managing chronic respiratory bacterial infections, which are resistant to traditional antibiotic treatments alone.

2D vanadium carbide MXenzyme to alleviate ROS-mediated inflammatory and neurodegenerative diseases

Reactive oxygen species (ROS) are generated and consumed in living organism for normal metabolism. Paradoxically, the overproduction and/or mismanagement of ROS have been involved in pathogenesis and progression of various human diseases. Here, we reported a two-dimensional (2D) vanadium carbide (V2C) MXene nanoenzyme (MXenzyme) that can mimic up to six naturally-occurring enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GPx), thiol peroxidase (TPx) and haloperoxidase (HPO). Based on these enzyme-mimicking properties, the constructed 2D V2C MXenzyme not only possesses high biocompatibility but also exhibits robust in vitro cytoprotection against oxidative stress. Importantly, 2D V2C MXenzyme rebuilds the redox homeostasis without perturbing the endogenous antioxidant status and relieves ROS-induced damage with benign in vivo therapeutic effects, as demonstrated in both inflammation and neurodegeneration animal models. These findings open an avenue to enable the use of MXenzyme as a remedial nanoplatform to treat ROS-mediated inflammatory and neurodegenerative diseases.

Arginine Decarboxylase Is Essential for Pneumococcal Stress Responses

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

Global transcriptional regulation by cell-free sup of S. Typhimurium peptide transporter mutant leads to inhibition of intraspecies biofilm initiation

Salmonella is a genus of widely spread Gram negative, facultative anaerobic bacteria, which is known to cause ¼th of the diarrhoeal morbidity and mortality globally. It causes typhoid fever and gastroenteritis by gaining access to the host gut through contaminated food and water. Salmonella utilizes its biofilm lifestyle to strongly resist antibiotics and persist in the host. Although the biofilm removal or dispersal have been studied widely, the inhibition of initiation Salmonella biofilm has not been studied much. This study was conducted to determine the anti-biofilm property of the cell-free supernatant obtained from a carbon-starvation inducible proline peptide transporter mutant (ΔyjiY) strain. Our study shows that Salmonella ΔyjiY culture supernatant inhibits biofilm initiation by regulating biofilm associated transcriptional network. This work demonstrates that highly abundant proteases like ecotin, HslV and GrpE cleave the protein aggregates, whereas global transcription regulators H-NS, FlgM regulate expression of SPIs and flagellar genes. Relatively low abundance of flavoredoxin, glutaredoxin, thiol peroxidase etc. leads to accumulation of ROS within the biofilm, and subsequent toxicity. This work further suggests that targeting these oxidative stress relieving proteins might be a good druggable choice to reduce Salmonella biofilm.

Global transcriptional regulation by cell-free supernatant of Salmonella Typhimurium peptide transporter mutant leads to inhibition of intra-species biofilm initiation

Salmonella is a genus of widely spread Gram negative, facultative anaerobic bacteria, which is known to cause ¼th of the diarrhoeal morbidity and mortality globally. It causes typhoid fever and gastroenteritis by gaining access to the host gut through contaminated food and water. Salmonella utilizes its biofilm lifestyle to strongly resist antibiotics and persist in the host. Although the biofilm removal or dispersal have been studied widely, the inhibition of initiation Salmonella biofilm has not been studied much. This study was conducted to determine the anti-biofilm property of the cell-free supernatant obtained from a carbon-starvation inducible proline peptide transporter mutant (ΔyjiY) strain. Our study shows that Salmonella ΔyjiY culture supernatant inhibits biofilm initiation by regulating biofilm associated transcriptional network. This work demonstrates that highly abundant proteases like ecotin, HslV and GrpE cleave the protein aggregates, whereas global transcription regulators H-NS, FlgM regulate expression of SPIs and flagellar genes. Relatively low abundance of flavoredoxin, glutaredoxin, thiol peroxidase etc. leads to accumulation of ROS within the biofilm, and subsequent toxicity. This work further suggests that targeting these oxidative stress relieving proteins might be a good druggable choice to reduce Salmonella biofilm.

Exploring the conformational transition between the fully folded and locally unfolded substates of Escherichia coli thiol peroxidase

Temporal acquisition of the fully folded conformational substate of the Escherichia coli thiol peroxidase by accelerated molecular dynamics simulations.

Exploring the Conformational Transition Between the Fully Folded and Locally Unfolded Substates of the Escherichia coli thiol peroxidase

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer in close to physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3 helices, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this crucial process, the mechanism of the FF→LU and the LU→FF transitions were studied using long time scale conventional molecular dynamic simulations and an enhanced conformational sampling technique for different oxidation and protonation states of CP and/or CR. Our results suggest that the FF→LU transition has a higher associated energy barrier than the refolding LU→FF process in agreement with the relatively slow experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of the αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionary optimized to fully speed-up the conformational transition of wild-type EcTPx.