Search

Mn2+ coordinated by orthophosphate (Pi), metabolites, or peptides acts as a superoxide dismutase (SOD), and these Mn antioxidant complexes are universally accumulated in extremely radiation-resistant cell types across the tree of life. This behavior prompted design of decapeptide DP1 (DEHGTAVMLK) as a Mn2+ ligand, and development of a highly potent Mn2+-antioxidant (MDP) containing [Pi] = 25 mM, and [DP1] = 3 mM, the ratio found in the radioresistant bacterium Deinococcus radiodurans, with [Mn2+] = 1 mM. MDP is an exceptional antioxidant, both in vitro and in vivo, and has reinvigorated the development of radiation-inactivated whole-cell vaccines. This study investigates the nature of the active Mn2+ complex in MDP. We measure the affinity of DP1 for the substitutionally labile Mn2+ ion using isothermal-titration calorimetry (ITC) and use changes in the Mn2+ solution EPR spectrum to determine affinities of Mn2+ for DP1 and for Pi, and to monitor Mn2+ ligation while titrated with the fixed Pi/DP1 ratio of MDP, 25/3, using ENDOR/ESEEM to characterize DP1 ligation to Mn2+. In parallel, 1H NMR of DP1 was used to monitor binding interactions between Pi and DP1, and DP1 binding to the diamagnetic Ca2+. We report: i) DP1 forms an extremely weak, dynamic Mn2+ complex (Ka ≈ 40 M-1) ii) Mn2+ binds Pi much more strongly (Ka ≈ 390 M-1) as shown previously, but iii) DP1 and Pi jointly bind to Mn2+ in MDP to form a ternary Mn2+ (Pi) (DP1) complex with greater formation-constant than Pi alone (Ka app ≈ 670 M-1). It is this ternary complex that is the superb antioxidant in MDP. [ABSTRACT FROM AUTHOR]

Despite their efficacy, the currently available polio vaccines, oral polio vaccine (OPV) and inactivated polio vaccine (IPV), possess inherent flaws posing significant challenges in the global eradication of polio. OPV, which uses live Sabin attenuated strains, carries the risk of reversion to pathogenic forms and causing vaccine-associated paralytic poliomyelitis (VAPP) and vaccine-derived polio disease (VDPD) in incompletely vaccinated or immune-compromised individuals. Conventional IPVs, which are non-replicative, are more expensive to manufacture and introduce biohazard and biosecurity risks due to the use of neuropathogenic strains in production. These types of limitations have led to a call by the Global Polio Eradication Initiative and others for the development of updated polio vaccines. We are developing a novel Ultraviolet-C radiation (UVC) inactivation method that preserves immunogenicity and is compatible with attenuated strains of polio. The method incorporates an antioxidant complex, manganese-decapeptide-phosphate (MDP), derived from the radioresistant bacterium Deinococcus radiodurans. The inclusion of MDP protects the immunogenic neutralizing epitopes from damage during UVC inactivation. The novel vaccine candidate, ultraIPVTM, produced using these methods demonstrates three crucial attributes: complete inactivation, which precludes the risk of vaccine-associated disease; use of non-pathogenic strains to reduce production risks; and significantly enhanced yield of doses per milligram of input virus, which could increase vaccine supply while reducing costs. Additionally, ultraIPVTM retains antigenicity post-freeze–thaw cycles, a testament to its robustness. [ABSTRACT FROM AUTHOR]

Networks of transcriptional and post-transcriptional regulators are critical for bacterial survival and adaptation to environmental stressors. While transcriptional regulators provide rapid activation and/or repression of a wide-network of genes, post-transcriptional regulators, such as small RNAs (sRNAs), are also important to fine-tune gene expression. However, the mechanisms of sRNAs remain poorly understood, especially in less-studied bacteria. Deinococcus radiodurans is a gram-positive bacterium resistant to extreme levels of ionizing radiation (IR). Although multiple unique regulatory systems (e.g., the Radiation and Desiccation Response (RDR)) have been identified in this organism, the role of post-transcriptional regulators has not been characterized within the IR response. In this study, we have characterized an sRNA, PprS (formerly Dsr2), as a post-transcriptional coordinator of IR recovery in D. radiodurans. PprS showed differential expression specifically under IR and knockdown of PprS resulted in reduced survival and growth under IR, suggesting its importance in regulating post-radiation recovery. We determined a number of potential RNA targets involved in several pathways including translation and DNA repair. Specifically, we confirmed that PprS binds within the coding region to stabilize the pprM (DR_0907) transcript, a RDR modulator. Overall, these results are the first to present an additional layer of sRNA-based control in DNA repair pathways associated with bacterial radioresistance. [ABSTRACT FROM AUTHOR]

The proper functioning of many proteins requires their transport to the correct cellular compartment or their secretion. Signal recognition particle (SRP) is a major protein transport pathway responsible for the co-translational movement of integral membrane proteins as well as periplasmic proteins. Deinococcus radiodurans is a ubiquitous bacterium that expresses a complex phenotype of extreme oxidative stress resistance, which depends on proteins involved in DNA repair, metabolism, gene regulation, and antioxidant defense. These proteins are located extracellularly or subcellularly, but the molecular mechanism of protein localization in D. radiodurans to manage oxidative stress response remains unexplored. In this study, we characterized the SRP complex in D. radiodurans R1 and showed that the knockdown (KD) of the SRP RNA (Qpr6) reduced bacterial survival under hydrogen peroxide and growth under chronic ionizing radiation. Through LC-mass spectrometry (MS/MS) analysis, we detected 162 proteins in the periplasm of wild-type D. radiodurans , of which the transport of 65 of these proteins to the periplasm was significantly reduced in the Qpr6 KD strain. Through Western blotting, we further demonstrated the localization of the catalases in D. radiodurans , DR_1998 (KatE1) and DR_A0259 (KatE2), in both the cytoplasm and periplasm, respectively, and showed that the accumulation of KatE1 and KatE2 in the periplasm was reduced in the SRP-defective strains. Collectively, this study establishes the importance of the SRP pathway in the survival and the transport of antioxidant proteins in D. radiodurans under oxidative stress. [ABSTRACT FROM AUTHOR]

A concerted action on the part of international agencies and national governments has resulted in the near-eradication of poliomyelitis. However, both the oral polio vaccine (OPV) and the inactivated polio vaccine (IPV) have deficiencies which make them suboptimal for use after global eradication. OPV is composed of attenuated Sabin strains and stimulates robust immunity, but may revert to neurovirulent forms in the intestine which can be shed and infect susceptible contacts. The majority of IPV products are manufactured using pathogenic strains inactivated with formalin. Upon eradication, the production of large quantities of pathogenic virus will present an increased biosecurity hazard. A logical ideal endgame vaccine would be an inactivated form of an attenuated strain that could afford protective immunity while safely producing larger numbers of doses per unit of virus stock than current vaccines. We report here the development of an ionizing radiation (IR)-inactivated Sabin-based vaccine using a reconstituted Mn-decapeptide (MDP) antioxidant complex derived from the radioresistant bacterium Deinococcus radiodurans. In bacteria, Mn2+-peptide antioxidants protect proteins from oxidative damage caused by extreme radiation exposure. Here we show for the first time, that MDP can protect immunogenic neutralizing epitopes in picornaviruses. MDP protects epitopes in Polio Virus 1 and 2 Sabin strains (PV1-S and PV2-S, respectively), but viral genomic RNA is not protected during supralethal irradiation. IR-inactivated Sabin viruses stimulated equivalent or improved neutralizing antibody responses in Wistar rats compared to the commercially used IPV products. Our approach reduces the biosecurity risk of the current PV vaccine production method by utilizing the Sabin strains instead of the wild type neurovirulent strains. Additionally, the IR-inactivation approach could provide a simpler, faster and less costly process for producing a more immunogenic IPV. Gamma-irradiation is a well-known method of virus inactivation and this vaccine approach could be adapted to any pathogen of interest. [ABSTRACT FROM AUTHOR]

The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of γ-rays (12,000 Gy), 20 times greater than Escherichia coli, is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) "antioxidant" Mn2+-metabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)-scavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn2+ speciation in D. radiodurans and E. coli cells and their responses to 10 kGy γ-irradiation. The Mn2+ of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn2+ of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn2+ speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn2+ complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn2+ speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation- induced ROS because it lacks an adequate supply of LMW Mn antioxidants. [ABSTRACT FROM AUTHOR]

Summary: Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn2+ complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25–40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist. [ABSTRACT FROM AUTHOR]

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn2+ and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn2+-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species. [ABSTRACT FROM AUTHOR]

In classical models of radiation toxicity, DNA is the molecule that is most affected by ionizing radiation (IR). However, recent data show that the amount of protein damage caused during irradiation of bacteria is better related to survival than to DNA damage. In this Opinion article, a new model is presented in which proteins are the most important target in the hierarchy of macromolecules affected by IR. A first line of defence against IR in extremely radiation-resistant bacteria might be the accumulation of manganese complexes, which can prevent the production of iron-dependent reactive oxygen species. This would allow an irradiated cell to protect sufficient enzymatic activity needed to repair DNA and survive. [ABSTRACT FROM AUTHOR]

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection. [ABSTRACT FROM AUTHOR]

A response by Michael J. Daly to a letter to the editor about his article "A new perspective on radiation resistance based on Deinococcus radiodurans" in the 2009 issue is presented.

The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of DNA repair systems and 40 Gy IR induces less than one double-strand break in its genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this organism's great sensitivity to IR. [ABSTRACT FROM AUTHOR]

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars. [ABSTRACT FROM AUTHOR]

Over the last ten years, Chikungunya virus (CHIKV), an Old World alphavirus has caused numerous outbreaks in Asian and European countries and the Americas, making it an emerging pathogen of great global health importance. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, on the other hand, has been developed as a bioweapon in the past due to its ease of preparation, aerosol dispersion and high lethality in aerosolized form. Currently, there are no FDA approved vaccines against these viruses. In this study, we used a novel approach to develop inactivated vaccines for VEEV and CHIKV by applying gamma-radiation together with a synthetic Mn-decapeptide-phosphate complex (MnDpPi), based on manganous-peptide-orthophosphate antioxidants accumulated in the extremely radiation-resistant bacterium Deinococcus radiodurans . Classical gamma-irradiated vaccine development approaches are limited by immunogenicity-loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus-inactivation. However, addition of MnDpPi during irradiation process selectively protects proteins, but not the nucleic acids, from the radiation-induced oxidative damage, as required for safe and efficacious vaccine development. Previously, this approach was used to develop a bacterial vaccine. In the present study, we show that this approach can successfully be applied to protecting mice against viral infections. Irradiation of VEEV and CHIKV in the presence of MnDpPi resulted in substantial epitope preservation even at supra-lethal doses of gamma-rays (50,000 Gy). Irradiated viruses were found to be completely inactivated and safe in vivo (neonatal mice). Upon immunization, VEEV inactivated in the presence of MnDpPi resulted in drastically improved protective efficacy. Thus, the MnDpPi-based gamma-inactivation approach described here can readily be applied to developing vaccines against any pathogen of interest in a fast and cost-effective manner. [ABSTRACT FROM AUTHOR]

The extremely radiation resistant bacterium, Deinococcus radiodurans, contains a spectrum of genes that encode for multiple activities that repair DNA damage. We have cloned and expressed the product of three predicted uracil-DNA glycosylases to determine their biochemical function. DR0689 is a homologue of the Escherichia coli uracil-DNA glycosylase, the product of the ung gene; this activity is able to remove uracil from a U:G and U:A base pair in double-stranded DNA and uracil from single-stranded DNA and is inhibited by the Ugi peptide. DR1751 is a member of the class 4 family of uracil-DNA glycosylases such as those found in the thermophiles Thermotoga maritima and Archaeoglobus fulgidus. DR1751 is also able to remove uracil from a U:G and U:A base pair; however, it is considerably more active on single-stranded DNA. Unlike its thermophilic relatives, the enzyme is not heat stable. Another putative enzyme, DR0022, did not demonstrate any appreciable uracil-DNA glycosylase activity. DR0689 appears to be the major activity in the organism based on inhibition studies with D. radiodurans crude cell extracts utilizing the Ugi peptide. The implications for D. radiodurans having multiple uracil-DNA glycosylase activities and other possible roles for these enzymes are discussed. [Copyright &y& Elsevier]