Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus)

Ferritin is a conserved iron binding protein existing ubiquitously in prokaryotes and eukaryotes. In this study, the gene encoding a ferritin M subunit homologue (SoFer1) was cloned from red drum (Sciaenops ocellatus) and analyzed at expression and functional levels. The open reading frame of SoFer1 is 531 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE) preserved in many ferritins. The deduced amino acid sequence of SoFer1 possesses both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Expression of SoFer1 was tissue specific and responded positively to experimental challenges with Gram-positive and Gram-negative fish pathogens. Treatment of red drum liver cells with iron, copper, and oxidant significantly upregulated the expression of SoFer1 in time-dependent manners. To further examine the potential role of SoFer1 in antioxidation, red drum liver cells transfected transiently with SoFer1 were prepared. Compared to control cells, SoFer1 transfectants exhibited reduced production of reactive oxygen species following H₂O₂ challenge. Finally, to examine the iron binding potential of SoFer1, SoFer1 was expressed in and purified from Escherichia coli as a recombinant protein. Iron-chelating analysis showed that purified recombinant SoFer1 was capable of iron binding. Taken together, these results suggest that SoFer1 is likely to be a functional ferritin involved in iron sequestration, host immune defence against bacterial infection, and antioxidation.     

Design and synthesis of 3-isoxazolidone derivatives as new Chlamydia trachomatis inhibitors

Chlamydia trachomatis (Ct) is a bacterial human pathogen responsible for the development of trachoma, the worldwide infection leading to blindness, and is also a major cause of sexually transmitted diseases. As iron is an essential metabolite for this bacterium, iron depletion presents a promising strategy to limit Ct proliferation. The aim of this study is to synthesize 3-isoxazolidone derivatives bearing known chelating moieties in an attempt to develop new bactericidal anti-Chlamydiaceae molecules. We have investigated the paths by which these new compounds affect Ct serovar L2 development in HeLa cells, in the presence or absence of exogenously added iron. The iron-chelating properties of these molecules were also determined. Our data reveal important bactericidal effects which are distinguishable from those due to iron chelation.     

Increased Gastric IL-1β Concentration and Iron Deficiency Parameters in H. pylori Infected Children

Association between H. pylori infection, iron deficiency and iron deficiency anaemia has been described, but the mechanisms involved have not been established. We hypothesized that in H. pylori infected children increased gastric concentrations of IL-1β and/or TNF-α, both potent inhibitors of gastric acid secretion that is essential for iron absorption, are predictors for low blood concentrations of ferritin and haemoglobin, markers of early depletion of iron stores and anaemia, respectively. We evaluated 125 children undergoing endoscopy to clarify the origin of gastrointestinal symptoms. Gastric specimens were obtained for H. pylori status and cytokine evaluation and blood samples for determination of iron deficiency/iron deficiency anaemia parameters and IL1 cluster and TNFA polymorphisms that are associated with increased cytokine secretions. Higher IL-1β and TNF-α gastric concentrations were observed in H. pylori-positive (n = 47) than in -negative (n = 78) children. Multiple linear regression models revealed gastric IL-1β, but not TNF-α, as a significant predictor of low ferritin and haemoglobin concentrations; results were reproduced in young children in whom IL1RN polymorphic genotypes associated with higher gastric IL-1β expression and lower blood ferritin and haemoglobin concentrations. In conclusion, high gastric levels of IL-1β can be the link between H. pylori infection and iron deficiency/iron deficiency anaemia in childhood.     

Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.     

Lipopolysaccharides of Rhizobium etli Strain G12 Act in Potato Roots as an Inducing Agent of Systemic Resistance to Infection by the Cyst Nematode Globodera pallida

Recent studies have shown that living and heat-killed cells of the rhizobacterium Rhizobium etli strain G12 induce in potato roots systemic resistance to infection by the potato cyst nematode Globodera pallida. To better understand the mechanisms of induced resistance, we focused on identifying the inducing agent. Since heat-stable bacterial surface carbohydrates such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) are essential for recognition in the symbiotic interaction between Rhizobium and legumes, their role in the R. etli-potato interaction was studied. EPS and LPS were extracted from bacterial cultures, applied to potato roots, and tested for activity as an inducer of plant resistance to the plant-parasitic nematode. Whereas EPS did not affect G. pallida infection, LPS reduced nematode infection significantly in concentrations as low as 1 and 0.1 mg ml⁻¹. Split-root experiments, guaranteeing a spatial separation of inducing agent and challenging pathogen, showed that soil treatments of one half of the root system with LPS resulted in a highly significant (up to 37%) systemic induced reduction of G. pallida infection of potato roots in the other half. The results clearly showed that LPS of R. etli G12 act as the inducing agent of systemic resistance in potato roots.     

Jumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is Involved in Defense-Related Gene Activation in Rice

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.     

Wolbachia-Associated Bacterial Protection in the Mosquito Aedes aegypti

Background

Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.

Methodology/Findings

Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction.

Conclusions/Significance

This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.     

Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.)

Priming plants by non-pathogenic bacteria allows the host to save energy and to reduce time needed for development of defense reaction during a pathogen attack. However, information on the role of endophytes in plant defense is limited. Here, the ability of endophytic bacteria to promote growth and resistance of potato plants towards infection by the necrotroph Pectobacterium atrosepticum was studied. A Pseudomonas sp. strain was selected due to antagonism towards bacterial pathogens and a Methylobacterium sp. strain because of efficient plant colonization. The aim of this study was to find if there is any correlation between plant growth promotion and induction of resistance by endophytes of potato, as well as to study the putative mechanisms of endophytes interacting with the plant during resistance induction. Both tested strains promoted growth of potato shoots but only the Pseudomonas sp. increased potato resistance towards the soft rot disease. Induction of disease resistance by the Methylobacterium sp. was inversely proportional to the size of bacterial population used for inoculation. The plant antioxidant system was moderately activated during the induction of resistance by the biocontrol strains. qPCR data on expression of marker genes of induced systemic resistance and acquired systemic resistance in endophyte-infected Arabidopsis plants showed activation of both salicylic acid and jasmonate/ethylene-dependent pathways after challenge inoculation with the pathogen. We suggest that some endophytes have the potential to activate both basal and inducible plant defense systems, whereas the growth promotion by biocontrol strains may not correlate with induction of disease resistance.     

Intracellular Survival and Persistence of Chlamydia muridarum Is Determined by Macrophage Polarization

Macrophages can display a number of distinct phenotypes, known collectively as polarized macrophages. The best defined of these phenotypes are the classically-activated, interferon gamma (IFNγ)/LPS induced (M1) and alternatively-activated, IL-4 induced (M2) macrophages. The goal of this study is to characterize macrophage- Chlamydia interactions in the context of macrophage polarization. Here we use Chlamydia muridarum and murine bone-marrow derived macrophages to show Chlamydia does not induce M2 polarization in macrophages as a survival strategy. Unexpectedly, the infection of macrophages was silent with no upregulation of M1 macrophage-associated genes. We further demonstrate that macrophages polarized prior to infection have a differential capacity to control Chlamydia . M1 macrophages harbor up to 40-fold lower inclusion forming units (IFU) than non-polarized or M2 polarized macrophages. Gene expression analysis showed an increase in 16sRNA in M2 macrophages with no change in M1 macrophages. Suppressed Chlamydia growth in M1 macrophages correlated with the induction of a bacterial gene expression profile typical of persistence as evident by increased Euo expression and decreased Omp1 and Tal expression. Observations of permissive Chlamydia growth in non-polarized and M2 macrophages and persistence in M1 macrophages were supported through electron microscopy. This work supports the importance of IFNγ in the innate immune response to Chlamydia . However, demonstration that the M1 macrophages, despite an antimicrobial signature, fail to eliminate intracellular Chlamydia supports the notion that host–pathogen co-evolution has yielded a pathogen that can evade cellular defenses against this pathogen, and persist for prolonged periods of time in the host.     

Overexpression of Os2H16 enhances resistance to phytopathogens and tolerance to drought stress in rice

Os2H16, a rice gene of unknown function, has been previously reported to be upregulated in response to infection by Xanthomonas oryzae pv. oryzae. In this study, expression patterns of Os2H16 were analyzed, demonstrating that expression of Os2H16 was dramatically induced by both bacterial and fungal infection as well as by drought stress, but repressed by salt treatment. To further investigate the role of Os2H16 in plant defense responses to abiotic and biotic stresses, transgenic lines of rice were developed. In comparison with wild-type rice, transgenic lines overexpressing Os2H16 show enhanced tolerance to bacterial blight and sheath blight disease, respectively caused by Xanthomonas oryzae pv. oryzae and Rhizoctonia solani. On the contrary, Os2H16 knockdown lines were more susceptible to both pathogens. Consistent with their individual phenotypes, upon inoculation, the expression of defense-related marker genes were elevated in Os2H16 overexpression individuals than in wild-type, while they were significantly reduced in Os2H16 knockdown lines. We also show that Os2H16 overexpression lines display enhanced tolerance to drought stress and elevated induction of drought-related genes, compared to wild-type rice. Os2H16 knockdown lines were more sensitive to drought stress and exhibited reduced induction of drought-related genes. Our study provides the first functional characterization of the rice Os2H16 gene, and suggests that Os2H16 positively modulate plant defense to abiotic and biotic stress.     

Cloning and Characterization of a Novel Drosophila Stress Induced DNase

Drosophila melanogaster flies mount an impressive immune response to a variety of pathogens with an efficient system comprised of both humoral and cellular responses. The fat body is the main producer of the anti-microbial peptides (AMPs) with anti-pathogen activity. During bacterial infection, an array of secreted peptidases, proteases and other enzymes are involved in the dissolution of debris generated by pathogen clearance. Although pathogen destruction should result in the release a large amount of nucleic acids, the mechanisms for its removal are still not known. In this report, we present the characterization of a nuclease gene that is induced not only by bacterial infection but also by oxidative stress. Expression of the identified protein has revealed that it encodes a potent nuclease that has been named Stress Induced DNase (SID). SID belongs to a family of evolutionarily conserved cation-dependent nucleases that degrade both single and double-stranded nucleic acids. Down-regulation of sid expression via RNA interference leads to significant reduction of fly viability after bacterial infection and oxidative stress. Our results indicate that SID protects flies from the toxic effects of excess DNA/RNA released by pathogen destruction and from oxidative damage.