Characterisation of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8, a powerful antagonist of fungal diseases of cucumber

Isolate 3.1T8 of Lysobacter enzymogenes (Christensen and Cook 1978), originating from the rhizosphere of cucumber and shown to have the potential to control Pythium aphanidermatum, is described. The strain produces extracellular proteases and lipases and shows high levels of resistance against streptomycin, kanamycin and tetracycline, but not to chloramphenicol. It shows strong in vitro antibiosis against P. aphanidermatum and several other phytopathogenic fungi. In order to identify the isolate, a carbon substrate oxidation profile (Biolog) was generated, and fatty acid methyl ester (FAME) analysis was performed. Also, the 16S rRNA gene was cloned and sequenced. With Biolog and FAME analysis, no assignment to species level was possible, because the species was not in the respective databases. BLAST analysis of the obtained sequence, followed by phylogenetic analysis, using a number of related and unrelated sequences, showed that the isolate was most closely related to Lysobacter enzymogenes (Christensen and Cook 1978).     

Immune Response in a Wild Bird Is Predicted by Oxidative Status,but Does Not Cause Oxidative Stress

The immune system provides vital protection against pathogens, but extensive evidence suggests that mounting immune responses can entail survival and fecundity costs. The physiological mechanisms that underpin these costs remain poorly understood, despite their potentially important role in shaping life-histories. Recent studies involving laboratory models highlight the possibility that oxidative stress could mediate these costs, as immune-activation can increase the production of reactive oxygen species leading to oxidative stress. However, this hypothesis has rarely been tested in free-ranging wild populations, where natural oxidative statuses and compensatory strategies may moderate immune responses and their impacts on oxidative status. Furthermore, the possibility that individuals scale their immune responses according to their oxidative status, conceivably to mitigate such costs, remains virtually unexplored. Here, we experimentally investigate the effects of a phytohaemagglutinin (PHA) immune-challenge on oxidative status in wild male and female white-browed sparrow weavers, Plocepasser mahali. We also establish whether baseline oxidative status prior to challenge predicts the scale of the immune responses. Contrary to previous work on captive animals, our findings suggest that PHA-induced immune-activation does not elicit oxidative stress. Compared with controls (n = 25 birds), PHA-injected birds (n = 27 birds) showed no evidence of a differential change in markers of oxidative damage or enzymatic and non-enzymatic antioxidant protection 24 hours after challenge. We did, however, find that the activity of a key antioxidant enzyme (superoxide dismutase, SOD) prior to immune-activation predicted the scale of the resulting swelling: birds with stronger initial SOD activity subsequently produced smaller swellings. Our findings (i) suggest that wild birds can mount immune responses without suffering from systemic oxidative stress, and (ii) lend support to biomedical evidence that baseline oxidative status can impact the scale of immune responses; a possibility not yet recognised in ecological studies of immunity.     

Parvulin (Par14), a Peptidyl-Prolyl cis-trans Isomerase, Is a Novel rRNA Processing Factor That Evolved in the Metazoan Lineage

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.Peptidyl-prolyl cis-trans isomerases (PPIases)¹ catalyze the rotation about the peptide bond on the amino-terminal side of proline, a step that can be rate-limiting for the folding of newly synthesized proteins (1). PPIases also have the ability to bind many proteins, thereby acting as chaperones; thus, they are believed to control the activity of proteins by regulating their folding, assembly, and intracellular trafficking (2–4). There are three families of PPIases, namely the cyclophilin (CyP), FK506-binding protein, and parvulin families. The CyP and FK506-binding protein families have been well established as targets of the immunosuppressants cyclosporin A and FK506, respectively (5–7).Together with Pin1, human parvulin (Par14, EPVH) constitutes the parvulin family and has been identified in all hitherto examined human tissues (8, 9). Par14 comprises 131 amino acid residues and has a 35-residue amino-terminal region that does not have sequence similarity to the WW domain (known to bind to phosphorylated serine/threonine-proline bonds in proteins and peptides) of Pin1. Phosphorylation at Ser-19 in this region regulates the subcellular localization and DNA binding activity of Par14; the phosphorylation is required for nuclear localization, and the dephosphorylation is a prerequisite for the binding of the first 25 residues to nuclear DNA (10). The 96-residue carboxyl-terminal domain has a 34.2% sequence identity with the PPIase domain of Pin1. Par14 reportedly has a substrate preference for positively charged residues preceding proline but not for phosphorylated Thr or Ser as is the case with Pin1; however, its rate constant for the prolyl cis to trans isomerization reaction is at least 1,000-fold lower than that of CyPs (9). NMR solution structural analysis has shown that Par14 folds into a βα3βαβ2 structure, which is essentially identical to that of Pin1 (11). The unstructured 35-residue amino-terminal region contains several basic residues and replaces the WW domain of Pin1 (11). This structural model explains the molecular basis for the preferential substrate specificity of Par14 for positively charged residues preceding proline as well as the putative role of the amino-terminal region as a DNA-binding domain. However, the physiological function of Par14 remains unknown.We previously reported that Par14 associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes as well as with many proteins that are implicated in the regulation of microtubule assembly or nucleolar reformation during mitosis (12, 13). We have proposed that Par14 is involved in ribosome biogenesis and/or nucleolar reassembly in mammalian cells during the pre- or postmitotic phases of the cell cycle. In the present study, we describe the comprehensive identification of protein components of the Par14-associated pre-rRNP complexes and establish Par14 as a de facto component of the pre-rRNP complexes in vivo. We also demonstrate that Par14 functions as a ribosomal RNA processing factor in mammalian ribosome biogenesis.     

Reproduction Is Associated with a Tissue-Dependent Reduction of Oxidative Stress in Eusocial Female Damaraland Mole-Rats (Fukomys damarensis)

Oxidative stress has been implicated as both a physiological cost of reproduction and a driving force on an animal''s lifespan. Since increased reproductive effort is generally linked with a reduction in survival, it has been proposed that oxidative stress may influence this relationship. Support for this hypothesis is inconsistent, but this may, in part, be due to the type of tissues that have been analyzed. In Damaraland mole-rats the sole reproducing female in the colony is also the longest lived. Therefore, if oxidative stress does impact the trade-off between reproduction and survival in general, this species may possess some form of enhanced defense. We assessed this relationship by comparing markers of oxidative damage (malondialdehyde, MDA; protein carbonyls, PC) and antioxidants (total antioxidant capacity, TAC; superoxide dismutase, SOD) in various tissues including plasma, erythrocytes, heart, liver, kidney and skeletal muscle between wild-caught reproductive and non-reproductive female Damaraland mole-rats. Reproductive females exhibited significantly lower levels of PC across all tissues, and lower levels of MDA in heart, kidney and liver relative to non-reproductive females. Levels of TAC and SOD did not differ significantly according to reproductive state. The reduction in oxidative damage in breeding females may be attributable to the unusual social structure of this species, as similar relationships have been observed between reproductive and non-reproductive eusocial insects.     

Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans,but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase

The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP⁺-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Δ strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance.The alveolar macrophage can produce microbicidal amounts of toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS) following phagocytosis (27, 53). Despite this, the opportunistic fungal pathogen Cryptococcus neoformans is able to inhabit and replicate within phagocytes of the mammalian host and to exit these cells unharmed (1, 2, 40). The intracellular pathogenicity of C. neoformans is most likely facilitated by stress resistance mechanisms, including a number of antioxidant proteins and pathways involved in the detoxification of ROS and RNS. Specifically, these include the synthesis of mannitol, a free radical scavenger (9, 20); the small protein flavohemoglobin denitrosylase (Fhb1), which is essential for resistance of C. neoformans to nitrosative stress (10, 14, 32); and the glutathione and thioredoxin antioxidant systems, which are both important for stress resistance and virulence (42, 43, 45).Even with different mechanisms of catalysis and/or cellular localization, one thing that these stress resistance proteins and pathways have in common is the requirement for NADPH as a cofactor. NADPH is used as an electron donor either in recycling of oxidized, inactive enzymes to reduced, active forms or directly in catalytic activity. For example, Fhb1 binds NADPH during its catalytic activity and uses it directly as an electron donor for the reduction of NO· to NO₃ (21). Catalases, which are highly conserved antioxidants that dismute H₂O₂ to molecular oxygen and water, consist of four units each with a molecule of NADPH bound in the core (18, 36, 59). The tripeptide glutathione (GSH) is oxidized to glutathione disulfide (GSSG), a homodimer held together by a disulfide bridge, during its oxidative state. GSSG can be reduced back to GSH by glutathione reductase, an enzyme that requires NADPH for electrons used in reduction. Similarly, glutathione peroxidase and thiol peroxidase ultimately depend on NADPH for recycling from an oxidized, inactive form back to a reduced, active form (57).NADPH is classically recognized as being produced by the highly conserved, cytosolic pentose phosphate pathway. This pathway has been shown to be important for reductive biochemistry during oxidative stress in many organisms. The pentose phosphate pathway is an essential factor in maintaining health of erythrocytes, cells that, due to their biological function, have considerable risk for oxidative damage. Humans deficient in the pathway have hemolytic anemia, as their erythrocytes are unable to maintain sufficient pools of reduced glutathione (68). Also, the pressure of oxidative stress can stimulate the pentose phosphate pathway. This has been shown in human lymphocytes (56); in the rat adrenal gland, liver, and pancreas (15, 16); and in bacteria (63).In fungi, the pentose phosphate pathway has been implicated in both oxidative stress resistance and adaptation to oxidative stress. In the model yeast Saccharomyces cerevisiae, NADPH-generating systems, including the pentose phosphate pathway, are critical for the ability of this organism to resist and adapt to high levels of oxidative stress (35, 47). It has also been shown that the cytosolic copper/zinc superoxide dismutase and the pentose phosphate pathway have overlapping roles in protecting S. cerevisiae from oxidative stress and that both systems are critical for maintaining the intracellular redox state (62). Furthermore, fungi may rely on the pentose phosphate pathway for more than reducing oxidative stress. Aspergillus nidulans requires a functional pentose phosphate pathway for nitrogen metabolism. Four A. nidulans mutants with independent defects in the pentose phosphate pathway were unable to grow on nitrite, nitrate, or various carbon sources, including 1% glucose, d-xylose, or d-glucoronate (28).The pathway has two phases, the oxidative phase and the nonoxidative phase. The oxidative phase consists of two successive oxidations and results in the production of NADPH. The first enzyme in the oxidative phase of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf). Zwf catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate and is highly specific for NADP⁺ as a cofactor (49, 67). There is abundant evidence supporting the role of Zwf in oxidative stress resistance. In addition to Zwf deficiency causing hemolytic anemia, Zwf has been also been implicated in maintenance of DNA repair systems during oxidative stress, as some cancers and aging disorders have also been linked to Zwf deficiency (30). For instance, Chinese hamster ovarian cells that are Zwf null have enhanced radiation sensitivity and a reduced ability to repair double-strand breaks due to the inactivation of Ku, a heterodimer DNA repair protein. In this case, the inactivation of Ku is the result of overoxidation of key cysteine residues on the protein due to the lack of sufficient reduced GSH (3). In the model yeast Saccharomyces cerevisiae, deletion of ZWF1 results in sensitivity to oxidative stress. ZWF1 is also important for the adaptive response to oxidative stress in S. cerevisiae. ZWF1-null mutants and wild-type cells were pretreated with 0.2 mM H₂O₂ and then challenged with 2 mM H₂O₂. While a large increase in tolerance to the high level of H₂O₂ was observed in the wild-type cells pretreated with 0.2 mM H₂O₂, the zwf1Δ strain was unable to tolerate the higher concentration (33). In Candida albicans, another pathogenic fungus, ZWF1 is upregulated during oxidative stress (38).Another source of NADPH is NADP⁺-dependent isocitrate dehydrogenase (Idp) (55), a ubiquitous enzyme that in systems ranging from humans to yeasts to plants has been found in the cytosol, peroxisomes, or mitochondria (12, 19, 70). Although this enzyme can be targeted to mitochondria, it is distinct from the NAD⁺-dependent isocitrate dehydrogenase (Idh) that functions in the mitochondria as part of the Krebs cycle. However, similarly to Idh, Idp catalyzes the decarboxylation of isocitrate to α-ketoglutarate (29). This reaction can be performed in the mitochondria, in the cytosol, or in peroxisomes using isocitrate formed from citrate exported across the mitochondrial membrane. This allows for the production of NADPH in cellular compartments without reliance of active transport of NADPH across membranes (11). It is important to have reductive power produced directly within organelles for protection from exogenous as well as endogenous stressor. For example, NADPH is consumed in peroxisomes by enzymes such as catalase and uric acid oxidase, that counteract the ROS produced during breakdown of lipids (4, 5, 31). Mitochondria particularly require reductive capability, as these organelles are susceptible to endogenous ROS produced during cellular respiration and also to exogenous RNS (52). The proteins that make up the electron transport chain are prone to damage by nitric oxide, peroxynitrite, and S-nitrosothiols (6). Nitric oxide and peroxynitrite have been shown to cause irreversible damage to cytochrome c reductase, NADH dehydrogenase, and the succinate-ubiquinone complex; the common mechanism of damage is sequestration of iron/sulfur centers of the proteins (54, 69). Thus, without a means of detoxification, the mitochondrial membrane loses potential and the ability to continue respiration, leading to death of the stressed cell. In C. neoformans, some antioxidant enzymes that are located at the mitochondria and dependent on NADPH for function include catalases, superoxide dismutases, cytochrome c peroxidase, and flavohemoglobin denitrosylase (7, 24, 25, 26). These enzymes are important for stress resistance or virulence of C. neoformans due to their role in high-temperature growth (24, 25) or nitrosative stress resistance (10, 14, 26).In humans, there is one IDP gene that results in mitochondrial and peroxisomal products (22). In S. cerevisiae, there are three IDP genes, which encode mitochondrial (IDP1), cytosolic (IDP2), and peroxisomal (IDP3) forms of the protein. Deletion of both ZWF1 and any one of the IDP genes in S. cerevisiae results in sensitivity to oxidative stress, likely due to a substantial decrease in NADPH produced in these double deletion mutants (41). In C. neoformans there is one predicted IDP gene (IDP1). Microarray data have indicated that this gene is upregulated 2.5-fold during nitrosative stress and thus may have a role in resistance to this stressor (44).Since so many factors essential for stress resistance in C. neoformans utilize NADPH, we hypothesize that the sources of this cofactor are likewise critical for stress resistance. Although Zwf1 is important for adaptation to oxidative stress in the fungi S. cerevisiae and C. albicans, we had previously found that C. neoformans is unable to adapt to oxidative stress (S. M. Brown and J. K. Lodge, unpublished data), and thus we had reason to suspect that the role of Zwf1 in C. neoformans may be different than that in other organisms. The role of Idp1 in stress resistance, especially in resistance to nitrosative stress, is relatively unknown. In this study we used biochemical and genetic approaches to compare the roles of Zwf1 and Idp1 in resistance to oxidative and nitrosative stress in C. neoformans. We found that the Zwf1 is dispensable for viability, for resistance to oxidative and nitrosative stress, and for NADPH production. In contrast, we found that Idp1 is important for resistance to nitrosative stress, specifically for maintaining healthy mitochondria during exposure to nitrosative stress.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

Relationship Between Sonic Hedgehog Protein, Brain-Derived Neurotrophic Factor and Oxidative Stress in Autism Spectrum Disorders

The etiology of autism spectrum disorders (ASD) is not well known but oxidative stress has been suggested to play a pathological role. We report here that the serum levels of Sonic hedgehog (SHH) protein and brain-derived neurotrophic factor (BDNF) might be linked to oxidative stress in ASD. By using the whole blood or polymorphonuclear leukocytes, we demonstrated that autistic children produced a significantly higher level of oxygen free radicals (OFR). In addition, we found significantly higher levels of serum SHH protein in children with mild as well as severe form of autism. We also found that the serum level of BDNF was significantly reduced in autistic children with mild form of the disorder but not with severe form of the disorder. Our findings are the first to report a correlation between SHH, BDNF and OFR in autistic children, suggesting a pathological role of oxidative stress and SHH in autism spectrum disorders.