Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment.

Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection and allow recognition of an original pathophysiologic mechanism underlying gastrointestinal diseases as well as identification of novel therapeutic targets.

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication.

Emerging antibiotic resistance in pathogenic bacteria like Mycobacterium sp., poses a threat to human health and therefore calls for the development of novel antibacterial strategies. We have recently discovered that bacterial membrane peptides, such as KdpF, possess anti-virulence properties when overproduced in pathogenic bacterial species. Overproduction of the KdpF peptide in Mycobacterium bovis BCG decreased bacterial replication within macrophages, without presenting antibacterial activity. We propose that KdpF functions as a regulatory molecule and interferes with bacterial virulence, potentially through interaction with the PDIM transporter MmpL7. We demonstrate here that KdpF overproduction in M. bovis BCG, increased bacterial susceptibility to nitrosative stress and thereby was responsible for lower replication rate within macrophages. Moreover, in a bacterial two-hybrid system, KdpF was able to interact not only with MmpL7 but also with two membrane proteins involved in nitrosative stress detoxification (NarI and NarK2), and a membrane protein of unknown function that is highly induced upon nitrosative stress (Rv2617c). Interestingly, we showed that the exogenous addition of KdpF synthetic peptide could affect the stability of proteins that interact with this peptide. Finally, the exogenous KdpF peptide presented similar biological effects as the endogenously expressed peptide including nitrosative stress susceptibility and reduced intramacrophage replication rate for M. bovis BCG. Taken together, our results establish a link between high levels of KdpF and nitrosative stress susceptibility to further highlight KdpF as a potent molecule with anti-virulence properties.

Role of Inflammation and Oxidative Stress in the Pathology of Ageing in COPD: Potential Therapeutic Interventions.

Ageing is defined as a progressive decline of homeostasis that occurs after the reproductive phase of life is complete, which is thought to arise because of impaired DNA repair following damage. This leads to an increased risk of disease or death. Ageing is one of the most important risk factors for most chronic diseases. Chronic obstructive pulmonary disease (COPD) represents an important component of the increasingly prevalent multiple chronic debilitating diseases that are a major cause of morbidity and mortality, particularly in the elderly. There is increasing evidence that the pathogenesis of COPD is linked to an accelerated ageing process. This review discusses the evidence supporting a number of mechanisms, including oxidative stress and ageing, in the pathogenesis of COPD. Greater understanding of these mechanisms leads to novel therapeutic interventions targeted at this heterogeneous disease.

[NO and cancer: itinerary of a double agent]. / Itinéraire d'un agent double - NO, S-nitrosylation et cancer.

Protein S-nitrosylation is now recognized as a ubiquitous regulatory mechanism. Like any post-translational modifications, S-nitrosylation is critical for the control of numerous cellular processes. It is now clear that S-nitrosylation is playing a double game, enhancing or inhibiting the tumor growth or the induction of cell death. Thanks to research aimed at demonstrating NO cytotoxic effects, new therapeutic strategies based on NO donor drugs have emerged. Although therapeutic NO donors can target a large number of proteins, the cellular mechanism is still not fully understood. This review reflects the current state of knowledge on S-nitrosylated proteins that take part of the oncogenic and apoptotic signaling, putting forward proteins with potential interest in cancer therapy.

A discrete role for FNR in the transcriptional response to moderate changes in oxygen by Haemophilus influenzae Rd KW20.

The survival by pathogenic bacteria within the specific conditions of an anatomical niche is critical for their persistence. These conditions include the combination of toxic chemicals, such as reactive oxygen (ROS) and reactive nitrogen species (RNS), with factors relevant to cell growth, such as oxygen. Haemophilus influenzae senses oxygen levels largely through the redox state of the intracellular fumarate-nitrate global regulator (FNR). H. influenzae certainly encounters oxygen levels that fluctuate, but in reality, these would rarely reach a state that results in FNR being fully reduced or oxidized. We were therefore interested in the response of H. influenzae to ROS and RNS at moderately high or low oxygen levels and the corresponding role of FNR. At these levels of oxygen, even though the growth rate of an H. influenzae fnr mutant was similar to wild type, its ROS and RNS tolerance was significantly different. Additionally, the subtle changes in oxygen did alter the whole cell transcriptional profile and this was different between the wild type and fnr mutant strains. It was the changed whole cell profile that impacted on ROS/RNS defence, but surprisingly, the FNR-regulated, anaerobic nitrite reductase (NrfA) continued to be expressed and had a role in this phenotype.

Plasma jet's shielding gas impact on bacterial inactivation.

One of the most desired aims in plasma medicine is to inactivate prokaryotic cells and leave eukaryotic cells unharmed or even stimulate proliferation to promote wound healing. The method of choice is to precisely control the plasma component composition. Here the authors investigate the inactivation of bacteria (Escherichia coli) by a plasma jet treatment. The reactive species composition created by the plasma in liquids is tuned by the use of a shielding gas device to achieve a reactive nitrogen species dominated condition or a reactive oxygen species dominated condition. A strong correlation between composition of the reactive components and the inactivation of the bacteria is observed. The authors compare the results to earlier investigations on eukaryotic cells and show that it is possible to find a plasma composition where bacterial inactivation is strongest and adverse effects on eukaryotic cells are minimized.

Global transcriptomic profiling of Schizosaccharomyces pombe in response to nitrosative stress.

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) cause nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. This is the first report on the global gene expression profile in response to NO in S. pombe using microarray. Among the 4824 genes reported for S. pombe, 818 were differentially expressed by at least 2-fold upon NO donor treatment. We previously showed that Pap1, the Activator Protein 1 transcription factor is required to combat nitrosative stress. In this study, the transcriptional response to NO in a null mutant for pap1 identified 45 genes that seem to be controlled by Pap1. Surprisingly, Pap1 regulated genes in S. pombe were distinctly different under nitrosative stress than those reported under oxidative stress. Genes of the pathway meiosis, cell cycle, spliceosome and oxidative phosphorylation were mostly affected under nitrosative stress in the fission yeast.

Oral streptococci and nitrite-mediated interference of Pseudomonas aeruginosa.

The oral cavity harbors a diverse community of microbes that are physiologically unique. Oral microbes that exist in this polymicrobial environment can be pathogenic or beneficial to the host. Numerous oral microbes contribute to the formation of dental caries and periodontitis; however, there is little understanding of the role these microbes play in systemic infections. There is mounting evidence that suggests that oral commensal streptococci are cocolonized with Pseudomonas aeruginosa during cystic fibrosis pulmonary infections and that the presence of these oral streptococci contributes to improved lung function. The goal of this study was to examine the underlying mechanism by which Streptococcus parasanguinis antagonizes pathogenic P. aeruginosa. In this study, we discovered that oral commensal streptococci, including Streptococcus parasanguinis, Streptococcus sanguinis, and Streptococcus gordonii, inhibit the growth of P. aeruginosa and that this inhibition is mediated by the presence of nitrite and the production of hydrogen peroxide (H2O2) by oral streptococci. The requirement of both H2O2 and nitrite for the inhibition of P. aeruginosa is due to the generation of reactive nitrogenous intermediates (RNI), including peroxynitrite. Transposon mutagenesis showed that a P. aeruginosa mutant defective in a putative ABC transporter permease is resistant to both streptococcus/nitrite- and peroxynitrite-mediated killing. Furthermore, S. parasanguinis protects Drosophila melanogaster from killing by P. aeruginosa in a nitrite-dependent manner. Our findings suggest that the combination of nitrite and H2O2 may represent a unique anti-infection strategy by oral streptococci during polymicrobial infections.

A short review on the implications of base excision repair pathway for neurons: relevance to neurodegenerative diseases.

Oxidative DNA damage results from the attack by reactive oxygen and nitrogen species (ROS/RNS) on human genome. This includes base modifications such as oxidized bases, abasic (AP) sites, and single-strand breaks (SSBs), all of which are repaired by the base excision repair (BER) pathway, one among the six known repair pathways. BER-pathway in mammalian cells involves several evolutionarily conserved proteins and is also linked to genome replication and transcription. The BER-pathway enzymes, namely, DNA glycosylases (DGs) and the end-processing proteins such as abasic endonuclease (APE1), form complexes with downstream repair enzymes via protein-protein and DNA-protein interactions. An emerging concept for BER proteins is their involvement in non-canonical functions associated to RNA metabolism, which is opening new interesting perspectives. Various mechanisms that are underlined in maintaining neuronal cell genome integrity are identified, but are inconclusive in providing protection against oxidative damage in neurodegenerative disorders, main emphasis is given towards the role played by the proteins of BER-pathway that is discussed. In addition, mechanisms of action of BER-pathway in nuclear vs. mitochondria as well as the non-canonical functions are discussed in connection to human neurodegenerative diseases.

Haemoglobins of Mycobacteria: structural features and biological functions.

The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.

The globins of cyanobacteria and algae.

Approximately, 20 years ago, a haemoglobin gene was identified within the genome of the cyanobacterium Nostoc commune. Haemoglobins have now been confirmed in multiple species of photosynthetic microbes beyond N. commune, and the diversity of these proteins has recently come under increased scrutiny. This chapter summarizes the state of knowledge concerning the phylogeny, physiology and chemistry of globins in cyanobacteria and green algae. Sequence information is by far the best developed and the most rapidly expanding aspect of the field. Structural and ligand-binding properties have been described for just a few proteins. Physiological data are available for even fewer. Although activities such as nitric oxide dioxygenation and oxygen scavenging are strong candidates for cellular function, dedicated studies will be required to complete the story on this intriguing and ancient group of proteins.

The globins of cold-adapted Pseudoalteromonas haloplanktis TAC125: from the structure to the physiological functions.

Evolution allowed Antarctic microorganisms to grow successfully under extreme conditions (low temperature and high O2 content), through a variety of structural and physiological adjustments in their genomes and development of programmed responses to strong oxidative and nitrosative stress. The availability of genomic sequences from an increasing number of cold-adapted species is providing insights to understand the molecular mechanisms underlying crucial physiological processes in polar organisms. The genome of Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct truncated globins exhibiting the 2/2 α-helical fold. One of these globins has been extensively characterised by spectroscopic analysis, kinetic measurements and computer simulation. The results indicate unique adaptive structural properties that enhance the overall flexibility of the protein, so that the structure appears to be resistant to pressure-induced stress. Recent results on a genomic mutant strain highlight the involvement of the cold-adapted globin in the protection against the stress induced by high O2 concentration. Moreover, the protein was shown to catalyse peroxynitrite isomerisation in vitro. In this review, we first summarise how cold temperatures affect the physiology of microorganisms and focus on the molecular mechanisms of cold adaptation revealed by recent biochemical and genetic studies. Next, since only in a very few cases the physiological role of truncated globins has been demonstrated, we also discuss the structural and functional features of the cold-adapted globin in an attempt to put into perspective what has been learnt about these proteins and their potential role in the biology of cold-adapted microorganisms.

Impact of nitrogen deposition at the species level.

In Europe and, increasingly, the rest of the world, the key policy tool for the control of air pollution is the critical load, a level of pollution below which there are no known significant harmful effects on the environment. Critical loads are used to map sensitive regions and habitats, permit individual polluting activities, and frame international negotiations on transboundary air pollution. Despite their fundamental importance in environmental science and policy, there has been no systematic attempt to verify a critical load with field survey data. Here, we use a large dataset of European grasslands along a gradient of nitrogen (N) deposition to show statistically significant declines in the abundance of species from the lowest level of N deposition at which it is possible to identify a change. Approximately 60% of species change points occur at or below the range of the currently established critical load. If this result is found more widely, the underlying principle of no harm in pollution policy may need to be modified to one of informed decisions on how much harm is acceptable. Our results highlight the importance of protecting currently unpolluted areas from new pollution sources, because we cannot rule out ecological impacts from even relatively small increases in reactive N deposition.

Host/microbe interactions revealed through "omics" in the symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri.

The association between Euprymna scolopes, the Hawaiian bobtail squid, and Vibrio fischeri, a bioluminescent bacterium, has served as a model for beneficial symbioses for over 25 years. The experimental tractability of this association has helped researchers characterize many of the colonization events necessary for symbiosis. Recent technological advances, such as the sequenced genome of V. fischeri, DNA microarrays, and high-throughput transcriptomics and proteomics, have allowed for the identification of host and symbiont factors that are important in establishing and maintaining specificity in the association. We highlight some of these findings pertaining to quorum sensing, luminescence, responses to reactive oxygen and nitrogen species, recognition of microbe-associated molecular patterns by the innate immune system of the host, and a diel rhythm that helps regulate the symbiont population. We also discuss how comparative genomics has allowed the identification of symbiont factors important for specificity and why sequencing the host's genome should be a priority for the research community.

Delivery method, target gene structure, and growth properties of target cells impact mutagenic responses to reactive nitrogen and oxygen species.

Dysregulated production of nitric oxide (NO•) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here, we compare cytotoxicity and mutagenicity induced by NO• and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system and a novel adaptation of a system for coculture. When exposed to preformed NO•, a cumulative dose of 620 µM min reduced the viability of TK6 cells at 24 h to 36% and increased mutation frequencies in the HPRT and TK1 genes to 7.7 × 10⁻6 (p < 0.05) and 24.8 × 10⁻6 (p < 0.01), 2.7- and 3.7-fold higher than background, respectively. In AS52 cells, cumulative doses of 1700 and 3700 µM min reduced viability to 49 and 22%, respectively, and increased the mutation frequency 10.2- and 14.6-fold higher than the argon control (132 × 10⁻6 and 190 × 10⁻6, respectively). These data show that TK6 cells were more sensitive than AS52 cells to killing by NO•. However, the two cell lines were very similar in relative susceptibility to mutagenesis; on the basis of fold increases in MF, average relative sensitivity values [(MF(exp)/MF(control))/cumulative NO• dose] were 5.16 × 10⁻³ and 4.97 × 10⁻³ µM⁻¹ min⁻¹ for TK6 cells and AS52 cells, respectively. When AS52 cells were exposed to reactive species generated by activated macrophages in the coculture system, cell killing was greatly reduced by the addition of NMA to the culture medium and was completely abrogated by combined additions of NMA and the superoxide scavenger Tiron, indicating the relative importance of NO• to loss of viability. Exposure in the coculture system for 48 h increased mutation frequency in the gpt gene by more than 9-fold, and NMA plus Tiron again completely prevented the response. Molecular analysis of gpt mutants induced by preformed NO• or by activated macrophages revealed that both doubled the frequency of gene inactivation (40% in induced vs 20% in spontaneous mutants). Sequencing showed that base-substitution mutations dominated the spectra, with transversions (30-40%) outnumbering transitions (10-20%). Virtually all mutations took place at guanine sites in the gene. G:C to T:A transversions accounted for about 30% of both spontaneous and induced mutations; G:C to A:T transitions amounted to 10-20% of mutants; insertions, small deletions, and multiple mutations were present at frequencies of 0-10%. Taken together, these results indicate that cell type and proximity to generator cells are critical determinants of cytotoxic and genotoxic responses induced by NO• and reactive species produced by activated macrophages.

Effects of nitric oxide on the survival and neuritogenesis of cerebellar Purkinje neurons.

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Secondary targets of nitrite-derived reactive nitrogen species: nitrosation/nitration pathways, antioxidant defense mechanisms and toxicological implications.

Nitrite, the primary metabolite of nitric oxide (NO) and a widely diffused component of human diet, plays distinct and increasingly appreciated roles in human physiology. However, when exposed to acidic environments, typically in the stomach, or under oxidative stress conditions, it may be converted to a range of reactive nitrogen species (RNS) which in turn can target a variety of biomolecules. Typical consequences of toxicological relevance include protein modification, DNA base deamination and the formation of N-nitrosamines, among the most potent mutagenic and carcinogenic compounds for humans. Besides primary biomolecules, nitrite can cause structural modifications to a variety of endogenous and exogenous organic compounds, ranging from polyunsaturated fatty acids to estrogens, tocopherol, catecholamines, furans, retinoids, dietary phenols, and a range of xenobiotics. The study of the interactions between nitrite and key food components, including phenolic antioxidants, has therefore emerged as an area of great promise for delineating innovative strategies in cancer chemoprevention. Depending on substrates and conditions, diverse reaction pathways may compete to determine product features and distribution patterns. These include nitrosation and nitration but also oxidation, via electron transfer to nitrosonium ion or nitrogen dioxide. This contribution aims to provide an overview of the main classes of compounds that can be targeted by nitrite and to discuss at chemical levels the possible reaction mechanisms under conditions that model those occurring in the stomach. The toxicological implications of the nitrite-modified molecules are finally addressed, and a rational chemical approach to the design of potent antinitrosing agents is illustrated.

Adaptation of Listeria monocytogenes to oxidative and nitrosative stress in IFN-γ-activated macrophages.

IFN-γ-activated macrophages are considered to be the primary effector cells in host defense against Listeria monocytogenes infections. However despite the induction of the complex host defense mechanisms, survival of L. monocytogenes in activated macrophages is still observed. Here we used a whole genome-based transcriptome approach to examine for bacterial genes specifically induced in IFN-γ-activated macrophages. We demonstrated that cells activated by IFN-γ had elevated oxidative and nitrosative stress levels in both the activated macrophages as well as in the intracellular replicating bacteria isolated from these infected cells. We found that a subset of 21 transcripts were specifically differentially regulated in bacteria growing in cells pretreated with IFN-γ. Bioinformatics and functional analysis revealed that many of these genes have roles involved in overcoming oxidative stress and contribute to bacterial survival within activated macrophages. We detected increased transcription of the putative trpE gene of L. monocytogenes, encoding an anthranilate synthase, in bacteria growing in IFN-γ cells indicating host cell metabolic restriction of bacterial growth. Indeed we found enhanced activation of host cell genes involved in the kynurenine pathway indicating an increased need of L. monocytogenes for tryptophan during replication in IFN-γ-activated macrophages.

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.