Erwinia amylovora catalases KatA and KatG are virulence factors and delay the starvation‐induced viable but non‐culturable (VBNC) response

The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H₂O₂) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H₂O₂‐neutralizing enzymes, such as catalases, during plant–pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non‐host environments was determined using catalase gene mutants and expression, as well as catalase activity analyses. The participation of E. amylovora exopolysaccharides (EPSs) in oxidative stress protection was also investigated. Our study revealed the following: (i) a different growth phase regulation of each catalase, with an induction by H₂O₂ and host tissues; (ii) the significant role of E. amylovora catalases as virulence and survival factors during plant–pathogen interactions; (iii) the induction of EPSs by H₂O₂ despite the fact that apparently they do not contribute to protection against this compound; and (iv) the participation of both catalases in the detoxification of the starvation‐induced intracellular oxidative stress, favouring the maintenance of culturability, and hence delaying the development of the viable but non‐culturable (VBNC) response.     

Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.     

Autocrine factors in defense of cytotoxic CTLL-2 cells from oxidative stress

The role of pyruvate and autocrine polypeptide factors (APF) secreted by cytotoxic IL-2-dependent CTLL-2 cells in cell defense from oxidative stress was investigated. The addition of a conditioned medium (CM) containing pyruvate and APF into CTLL-2 cell cultures significantly increased the cell survival under oxidative stress conditions induced by hydrogen peroxide (H₂O₂). The kinetics of (H₂O₂) removal from cell cultures with added CM has been registered. It has been shown that, at the beginning of oxidative stress (less than 15 min), H₂O₂ was mostly removed by means of its reaction with pyruvate contained in CM. Pyruvate content in CM was estimated as 138 ± 7 μM. Gel filtration on a column with Bio-Gel P-10 was used to eliminate pyruvate from CM. Gel filtration resulted in three CM fractions (A, B, and C) corresponding to three chromatogram peaks. Pyruvate was not detected in any fraction. The fraction A was the first to be eluted from the column and contained the largest molecules. In the cell survival test, fraction B had the highest protective ability for CTLL-2 cells under oxidative stress. Fraction A supported cell survival to a lesser degree and fraction C did not show any protective abilities. Fraction B added to cells under oxidative stress kept intracellular ATP content at a significantly higher level then in control cells. Moreover, it was found that APF from fraction B was able to react with H₂O₂ directly and inactivate it in the absence of cells. APF from fraction A did not have such properties.     

KatG,the Bifunctional Catalase of Xanthomonas citri subsp. citri,Responds to Hydrogen Peroxide and Contributes to Epiphytic Survival on Citrus Leaves

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This bacterium is exposed to reactive oxygen species (ROS) at different points during its life cycle, including those normally produced by aerobic respiration or upon exposition to ultraviolet (UV) radiation. Moreover, ROS are key components of the host immune response. Among enzymatic ROS-detoxifying mechanisms, catalases eliminate H₂O₂, avoiding the potential damage caused by this specie. Xcc genome includes four catalase genes. In this work, we studied the physiological role of KatG, the only bifunctional catalase of Xcc, through the construction and characterization of a modified strain (XcckatG), carrying an insertional mutation in the katG gene. First, we evaluated the involvement of KatG in the bacterial adaptive response to H₂O₂. XcckatG cultures exhibited lower catalase activity than those of the wild-type strain, and this activity was not induced upon treatment with sub-lethal doses of H₂O₂. Moreover, the KatG-deficient mutant exhibited decreased tolerance to H₂O₂ toxicity compared to wild-type cells and accumulated high intracellular levels of peroxides upon exposure to sub-lethal concentrations of H₂O₂. To further study the role of KatG in Xcc physiology, we evaluated bacterial survival upon exposure to UV-A or UV-B radiation. In both conditions, XcckatG showed a high mortality in comparison to Xcc wild-type. Finally, we studied the development of bacterial biofilms. While structured biofilms were observed for the Xcc wild-type, the development of these structures was impaired for XcckatG. Based on these results, we demonstrated that KatG is responsible for Xcc adaptive response to H₂O₂ and a key component of the bacterial response to oxidative stress. Moreover, this enzyme plays an important role during Xcc epiphytic survival, being essential for biofilm formation and UV resistance.     

Overexpression of a peroxiredoxin gene from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, <Emphasis Type="Italic">ThPrx1</Emphasis>, confers tolerance to oxidative stress in yeast and Arabidopsis

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H₂O₂) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H₂O₂ and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H₂O₂, superoxide (O₂^??) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.     

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H₂O₂) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.     

Cortisol promotes stress tolerance via DAF-16 in Caenorhabditis elegans

In this study, we studied the effects of cortisol and cortisone on the age-related decrease in locomotion in the nematode Caenorhabditis elegans and on the tolerance to heat stress at 35 °C and to oxidative stress induced by the exposure to 0.1% H₂O₂. Changes in mRNA expression levels of C. elegans genes related to stress tolerance were also analyzed. Cortisol treatment restored nematode movement following heat stress and increased viability under oxidative stress, but also shortened worm lifespan. Cortisone, a cortisol precursor, also restored movement after heat stress. Additionally, cortisol treatment increased mRNA expression of the hsp-12.6 and sod-3 genes. Furthermore, cortisol treatment failed to restore movement of daf-16-deficient mutants after heat stress, whereas cortisone failed to restore the movement of dhs-30-deficient mutants after heat stress. In conclusion, the results suggested that cortisol promoted stress tolerance via DAF-16 but shortened the lifespan, whereas cortisone promoted stress tolerance via DHS-30.     

The role of Acanthamoeba castellanii (T4 genotype) antioxidant enzymes in parasite survival under H2O2-induced oxidative stress

Acanthamoeba castellanii (A. castellanii) is an important opportunistic parasite. Induction of oxidative stress by the host immune system is one of the most important defense strategies against parasites. Hence, parasites partly deal with oxidative stress by different mechanisms. Identifying resistance mechanisms of A. castellanii parasites against oxidative stress is important to achieve a new therapeutic approach. Thus, this study aimed to understand the resistance mechanisms of A. castellanii, against oxidative stress. Trophozoites of A. castellanii were treated with different concentrations of H₂O₂. The half maximal inhibitory concentration (IC₅₀) of H₂O₂ was determined using the MTT assay. The induction of oxidative stress was confirmed by flow cytometer. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. The gene expression levels of CAT and SOD were measured by qRT-PCR. Furthermore, 3-amino-1:2:4-triazole (3-AT) and potassium cyanide (KCN) were used as specific inhibitors of CAT and SOD, respectively. Cell cycle assay and the apoptosis were evaluated by flow cytometer. The activities of SOD, CAT, GR, and GPx, showed an increase in oxidative stress. The cell cycle analysis revealed that most of the cellular population was in G0 and G1 phases. The apoptosis increased in oxidative stress conditions. Moreover, the apoptosis significantly increased after the specific inhibition of CAT and SOD under oxidative stress. The gene expression levels of CAT and SOD significantly increased under oxidative stress. A. castellanii can resist the host immune system through various mechanisms, including evoking its antioxidant enzymes. Therefore, by reducing or inhibiting the activity of the parasite's antioxidant enzymes such as SOD and CAT, it is possible to cope with A. castellanii.     

Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Also Exhibit Chaperone like Activity In-Vitro and In-Vivo

Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. Mycobacterium tuberculosis (M.tb) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases. We show that recombinant rPpiA and rPpiB can bind to non-native proteins in vitro and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography.E. coli cells overexpressing PpiA and PpiB of M.tb could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing M.tb PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions generated after treatment with H₂O₂ and CoCl₂ thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these M.tuberculosis cyclophilins may possibly function as a stress responder and consequently contribute to virulence.     

Human polynucleotide phosphorylase reduces oxidative RNA damage and protects HeLa cell against oxidative stress

We examined HeLa cell viability and RNA oxidative damage in response to hydrogen peroxide (H₂O₂) treatment. The level of damaged RNA, measured by the content of 8-hydroxyguanosine (7,8-dihydro-8-oxoguanosine, 8-oxoG), increases depending on H₂O₂ dosage and is inversely correlated with cell viability. The elevated level of 8-oxoG in RNA decreases after removal of oxidative challenge, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. Human polynucleotide phosphorylase (hPNPase), an exoribonuclease primarily located in mitochondria, has been previously shown to bind 8-oxoG-RNA with high affinity. The role of hPNPase in HeLa cell under oxidative stress conditions is examined here. Overexpression of hPNPase reduces RNA oxidation and increases cell viability against H₂O₂ insult. Conversely, hPNPase knockdown decreases viability and increases 8-oxoG level in HeLa cell exposed to H₂O₂. Our results suggest that hPNPase plays an important role in protecting cells and limiting damaged RNA under oxidative stress.     

CBS-derived H2S facilitates host colonization of Vibrio cholerae by promoting the iron-dependent catalase activity of KatB

Sensing and resisting oxidative stress is critical for Vibrio cholerae to survive in either the aquatic environment or the gastrointestinal tract. Previous studies mainly focused on the mechanisms of oxidative stress response regulation that rely on enzymatic antioxidant systems, while functions of non-enzymatic antioxidants are rarely discussed in V. cholerae. For the first time, we investigated the role of hydrogen sulfide (H₂S), the simplest thiol compound, in protecting V. cholerae against oxidative stress. We found that degradation of L-cysteine by putative cystathionine β-synthase (CBS) is the major source of endogenous H₂S in V. cholerae. Our results indicate that intracellular H₂S level has a positive correlation with cbs expression, while the enhanced H₂S production can render V. cholerae cells less susceptible to H₂O₂ in vitro. Using proteome analysis and real-time qPCR assay, we found that cbs expression could stimulate the expression of several enzymatic antioxidants, including reactive oxygen species (ROS) detoxifying enzymes SodB, KatG and AhpC, the DNA protective protein DPS and the protein redox regulator Trx1. Assays of ROS detoxification capacities revealed that CBS-derived H₂S could promote catalase activity at the post-translational level, especially for KatB, which serves as an important way that endogenous H₂S participates in H₂O₂ detoxification. The enhancement of catalase activity by H₂S is achieved through facilitating the uptake of iron. Adult mice experiments showed that cbs mutant has colonization defect, while either complementation of cbs or exogenous supplement of N-Acetyl-L-Cysteine restores its fitness in the host environment. Herein, we proposed that V. cholerae regulates CBS-dependent H₂S production for better survival and proliferation under ROS stress.     

Role of LytF and AtlS in eDNA Release by Streptococcus gordonii

Extracellular DNA (eDNA) is an important component of the biofilm matrix produced by many bacteria. In general, the release of eDNA is associated with the activity of muralytic enzymes leading to obvious cell lysis. In the Gram-positive oral commensal Streptococcus gordonii, eDNA release is dependent on pyruvate oxidase generated hydrogen peroxide (H₂O₂). Addition of H₂O₂ to cells grown under conditions non-permissive for H₂O₂ production causes eDNA release. Furthermore, eDNA release is maximal under aerobic growth conditions known to induce pyruvate oxidase gene expression and H₂O₂ production. Obvious cell lysis, however, does not occur. Two enzymes have been recently associated with eDNA release in S. gordonii. The autolysin AtlS and the competence regulated murein hydrolase LytF. In the present report, we investigated the role of both proteins in the H₂O₂ dependent eDNA release process. Single and double mutants in the respective genes for LytF and AtlS released less eDNA under normal growth conditions, but the AtlS mutant was still inducible for eDNA release by external H₂O₂. Moreover, we showed that the AtlS mutation interfered with the ability of S. gordonii to produce eDNA release inducing amounts of H₂O₂. Our data support a role of LytF in the H₂O₂ eDNA dependent release of S. gordonii as part of the competence stress pathway responding to oxidative stress.     

Overlapping and Complementary Oxidative Stress Defense Mechanisms in Nontypeable Haemophilus influenzae

The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H₂O₂), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX, and dps. The hktE mutant and a pgdX hktE double mutant were more sensitive than the parent to killing by H₂O₂. Conversely, the pgdX mutant was more resistant to H₂O₂ due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H₂O₂-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.     

Responses of marine macroalgae to hydrogen-peroxide stress

In this study, we determined the antioxidative potential of 15 marine macroalgae by measuring the photosynthetic efficiency under artificial oxidative stress after a 30-min exposure to a series of ascending H₂O₂ concentrations. Species exhibiting high maximum quantum yields (F_v/F_m values) were regarded as not susceptible towards H₂O₂ stress. In addition to the short-term stress experiments, the antioxidative defense systems (enzymatic and non-enzymatic) of selected algal species under longer exposure times to H₂O₂ were investigated.Species with striking photosynthetic activity under H₂O₂ stress were Chaetomorpha melagonium (Chlorophyta), showing 40% reduced F_v/F_m as compared to the control after 8 days of exposure to 20 mM H₂O₂. In Fucus distichus (Phaeophyta) F_v/F_m decreased to 50% of the control under the same exposure conditions. Polysiphonia arctica (Rhodophyta) exhibited highest F_v/F_m values with a reduction of only 25%, therefore possessing the highest antioxidative potential of the investigated species.In P. arctica the activities of the antioxidative enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as the pool size of the antioxidant ascorbic acid were investigated. When exposed to different H₂O₂ concentrations (0-2 mM) over 6 days, the intrinsic activities of SOD and GR were stimulated. In a kinetic study over 8 days, the activity of antioxidative enzymes APX and CAT as well as ascorbic acid content were recorded. APX activity was much higher in H₂O₂-treated thalli at the end of the experiment than in the control, also CAT activity increased significantly with increasing H₂O₂ stress. In parallel, ascorbic acid content was reduced under high H₂O₂ concentrations. Furthermore, by using GC-MS techniques in P. arctica bromophenolic compounds with antioxidative properties were identified.This study shows that the measurement of the in vivo fluorescence of photosystem II is a suitable tool to determine the effect of oxidative stress on macroalgae. From these studies it is obvious that different algal species have varying strategies against oxidative stress which correlate with zonation on the shore.     

The functional group on (E)-4,4′–disubstituted stilbenes influences toxicity and antioxidative activity in differentiated PC-12 cells

This work probes the relationship between stilbene functional group and biological activity. The biological activity of synthesized stilbenes (E)-4,4′-dicyanostilbene, (E)-4,4′-diacetylstilbene, (E)-4,4′-diaminostilbene, a novel stilbene, 1,1′-(vinylenedi-p-phenylene)diethanol, and (E)-stilbene was assessed at biologically relevant nanomolar concentrations using the MTS cell viability assay in differentiated PC-12 cells under optimal culture conditions and conditions of oxidative stress. Under optimal culture conditions the synthesized stilbene derivatives were found to be non-toxic to cells at concentrations up to 10 μg/ml. To mimic oxidative stress, the activity of these stilbene derivatives in the presence of 0.03% H₂O₂ was investigated. Stilbene derivatives with electron-withdrawing functional groups were 2–3 times more toxic than the H₂O₂ control, indicating that they may form toxic metabolites in the presence of H₂O₂. Fluorescence data supported that stilbene derivatives with electron-withdrawing functional groups, (E)-4,4′-dicyanostilbene and (E)-4,4′-diacetylstilbene, may react with H₂O₂. In contrast, the stilbene derivative with a strong electron-donating functional group, (E)-4,4′-diaminostilbene, rescued neurons from H₂O₂-induced toxicity. The DPPH assay confirmed that (E)-4,4′-diaminostilbene is able to scavenge free radicals. These data indicate that the Hammett value of the functional group correlates with the biological activity of (E)-4,4′-disubstituted stilbenes in differentiated PC-12 cells.