Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel β-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.

The bZIP transcription factor Rca1p is a central regulator of a novel CO₂ sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea.

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Neutrophils in infectious diseases

Host defense to intracellular pathogens depends upon both innate and adaptive cell-mediated immune responses. Polymorphonuclear neutrophil leukocytes which belong to the innate immune system are the first cells that are recruited massively within hours of microbial infection. Neutrophils are the main players in the killing of microorganisms and recently new methods of killing including nets formation have been described. Neutrophils mediate tissue damage at infected sites. By promoting tissue injury neutrophils contribute to the initiation of inflammation, which is now recognized as an essential step in launching immunity. The importance of neutrophils as decision shaper in the development of an immune response is only emerging as they have long been considered by immunologists as short lived, non-dividing cells, of poor interest. Now, neutrophils are emerging as key components of the inflammatory response, and are shown to have immunoregulatory roles in microbial infections. In addition, neutrophils were also reported to contribute to the recruitment and activation of antigen presenting cells. Thus early interactions between neutrophils and surrounding cells may influence the development/resolution of both inflammatory lesion and pathogen-specific immune response. The impact of neutrophils on cells present at the site of infection are only beginning to be studied and deserves more attention.In this e-book the reader will find updated information about the role of neutrophils in the pathogenesis of 1) bacterial diseases including sepsis, mycobacteria and Chlamydia infections, and of 2) parasitic diseases including leishmaniasis and toxoplasmosis. The role of neutrophils in the protection against microorganisms has largely been underestimated and, until recently, their role was mostly thought to limited to a "kill and die" response. New neutrophil mode of killing, such as their release of extracellular traps to kill extracellular bacterial pathogens, together with several microbial strategies designed to escape NETs are presented in Chapter 1. We will emphasize standard and advanced light microscopy techniques that allowed major advances in the understanding of neutrophil biology, through the visualization of the interaction of selected pathogens with neutrophils in living animals (Chapter 2).The aim of this e-book is to provide an overview of the recent advances made in the field of neutrophil biology. It will provide a basis for understanding future development that will occur in this area, and provide the reader with a short overview of some of the exciting new directions in which neutrophil research is moving.

Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection.

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

Human alveolar macrophages infected by virulent bacteria expressing SipB are a major source of active interleukin-18.

Recent publications have demonstrated that the protease caspase-1 is responsible for the processing of pro-interleukin 18 (IL-18) into the active form. Studies on cell lines and murine macrophages have shown that the bacterial invasion factor SipB activates caspase-1, triggering cell death. Thus, we investigated the role of SipB in the activation and release of IL-18 in human alveolar macrophages (AM), which are the first line of defense against inhaled pathogens. Under steady-state conditions, AM are a more important source of IL-18 than are dendritic cells (DC) and monocytes. Cytokine production by AM and DC was compared after both types of cells had been infected with a virulent strain of Salmonella enterica serovar Typhimurium and an isogenic sipB mutant, which were used as an infection model. Infection with virulent Salmonella led to marked cell death with features of apoptosis while both intracellular activation and release of IL-18 were demonstrated. In contrast, the sipB mutant did not induce such cell death or the release of active IL-18. The specific caspase-1 inhibitor Ac-YVAD-CMK blocked the early IL-18 release in AM infected with the virulent strain. However, the type of Salmonella infection did not differentially regulate IL-18 gene expression. We concluded that the bacterial virulence factor SipB plays an essential posttranslational role in the intracellular activation of IL-18 and the release of the cytokine in human AM.

Raa'an siipikarjanlihan kontaminoituminen patogeenisilla bakteereilla : kirjallisuuskatsaus

Summary: Contamination of raw poultry with pathogens - a literature review

Chemical and microbiological attributes of an oxisol treated with successive applications of sewage sludge¹

Studies on sewage sludge (SS) have confirmed the possibilities of using this waste as fertilizer and/or soil conditioner in crop production areas. Despite restrictions with regard to the levels of potentially toxic elements (PTE) and pathogens, it is believed that properly treated SS with low PTE levels, applied to soil at adequate rates, may improve the soil chemical and microbiological properties. This study consisted of a long-term field experiment conducted on a Typic Haplorthox (eutroferric Red Latosol) treated with SS for seven successive years for maize production, to evaluate changes in the soil chemical and microbiological properties. The treatments consisted of two SS rates (single and double dose of the crop N requirement) and a mineral fertilizer treatment. Soil was sampled in the 0-0.20 m layer and analyzed for chemical properties (organic C, pH, P, K, Ca, Mg, CEC, B, Cu, Fe, Mn, Zn, Cd, Ni, and Pb) and microbiological properties (basal respiration, microbial biomass activity, microbial biomass C, metabolic quotient, microbial quotient, and protease and dehydrogenase enzyme activities). Successive SS applications to soil increased the macro- and micronutrient availability, but the highest SS dose reduced the soil pH significantly, indicating a need for periodic corrections. The SS treatments also affected soil microbial activity and biomass negatively. There were no significant differences among treatments for maize grain yield. After seven annual applications of the recommended sludge rate, the heavy metal levels in the soil had not reached toxic levels.

Systemic antibodies can inhibit mouse mammary tumor virus-driven superantigen response in mucosa-associated lymphoid tissues.

Many mucosal pathogens invade the host by initially infecting the organized mucosa-associated lymphoid tissue (o-MALT) such as Peyer's patches or nasal cavity-associated lymphoid tissue (NALT) before spreading systemically. There is no clear demonstration that serum antibodies can prevent infections in o-MALT. We have tested this possibility by using the mouse mammary tumor virus (MMTV) as a model system. In peripheral lymph nodes or in Peyer's patches or NALT, MMTV initially infects B lymphocytes, which as a consequence express a superantigen (SAg) activity. The SAg molecule induces the local activation of a subset of T cells within 6 days after MMTV infection. We report that similar levels of anti-SAg antibody (immunoglobulin G) in serum were potent inhibitors of the SAg-induced T-cell response both in peripheral lymph nodes and in Peyer's patches or NALT. This result clearly demonstrates that systemic antibodies can gain access to Peyer's patches or NALT.

Polymorphisms in tumor necrosis factor-α increase susceptibility to intra-abdominal Candida infection in high-risk surgical ICU patients*.

OBJECTIVES: To evaluate the influence of genetic polymorphisms on the susceptibility to Candida colonization and intra-abdominal candidiasis, a blood culture-negative life-threatening infection in high-risk surgical ICU patients. DESIGN: Prospective observational cohort study. SETTING: Surgical ICUs from two University hospitals of the Fungal Infection Network of Switzerland. PATIENTS: Eighty-nine patients at high risk for intra-abdominal candidiasis (68 with recurrent gastrointestinal perforation and 21 with acute necrotizing pancreatitis). MEASUREMENTS AND MAIN RESULTS: Eighteen single-nucleotide polymorphisms in 16 genes previously associated with development of fungal infections were analyzed from patient's DNA by using an Illumina Veracode genotyping platform. Candida colonization was defined by recovery of Candida species from at least one nonsterile site by twice weekly monitoring of cultures from oropharynx, stools, urine, skin, and/or respiratory tract. A corrected colonization index greater than or equal to 0.4 defined "heavy" colonization. Intra-abdominal candidiasis was defined by the presence of clinical symptoms and signs of peritonitis or intra-abdominal abscess and isolation of Candida species either in pure or mixed culture from intraoperatively collected abdominal samples. Single-nucleotide polymorphisms in three innate immune genes were associated with development of a Candida corrected colonization index greater than or equal to 0.4 (Toll-like receptor rs4986790, hazard ratio = 3.39; 95% CI, 1.45-7.93; p = 0.005) or occurrence of intra-abdominal candidiasis (tumor necrosis factor-α rs1800629, hazard ratio = 4.31; 95% CI, 1.85-10.1; p= 0.0007; β-defensin 1 rs1800972, hazard ratio = 3.21; 95% CI, 1.36-7.59; p = 0.008). CONCLUSION: We report a strong association between the promoter rs1800629 single-nucleotide polymorphism in tumor necrosis factor-α and an increased susceptibility to intra-abdominal candidiasis in a homogenous prospective cohort of high-risk surgical ICU patients. This finding highlights the relevance of the tumor necrosis factor-α functional polymorphism in immune response to fungal pathogens. Immunogenetic profiling in patients at clinical high risk followed by targeted antifungal interventions may improve the prevention or preemptive management of this life-threatening infection.

Functional analysis of altered channel-activating protease-1 (CAP1) expression in the skin

Summary : The skin is a complex organ that protects the body against entry of pathogens and supplies a relatively dry and impermeable barrier to water loss. This barrier function is mainly provided by the epidermis, which is the outermost layer of the skin. Serine proteases are involved in skin physiology and it is known that mutations or alterations in their expression can lead to skin diseases. In order to investigate the importance of the regulated expression of CAPI/Prss8, a membrane bound serine protease expressed in the epidermis, we developed transgenic mice ectopically expressing CAPI/Prss8 in the skin. These animals exhibited a phenotype characterized by scaly skin, epidermal hypertrophy, inflammation and scratching behavior. This phenotype could be completely abolished in mice lacking the proteinase activated receptor 2 (PAR2) revealing PAR2 as a potential in vivo downstream target of CAP 1 /Prss8. We could also provide evidence of a CAP1 /Prss8 function independent of its catalytic activity. Additionally, mice ectopically expressing PAR2 in the skin developed a skin phenotype very similar to CAPI/Prss8 transgenic animals, supporting the hypothesis of PAR2 activation by CAPI/Prss8. We could furthermore demonstrate an inhibitory effect of the serine protease inhibitor nexin-I on CAPI/Prss8, since nexin-1 transgenic expression negated the skin phenotype observed in CAPI/Prss8 transgenic mice. CAP1/Prss8 and PAR2 transgenic animals, and the understanding of the interaction between CAPl/Prss8 and PAR2, can be helpful in developing potential CAPI/Prss8 and PAR2 inhibitory molecules that may be used as drugs to treat ichthyoses-like skin diseases. Résumé : La peau est un organe complexe qui protège le corps contre l'entrée des pathogènes et forme une barrière imperméable qui empêche la déshydratation. Cette fonction de barrière est surtout fournie par l'épiderme, la couche la plus superficielle de la peau. Le bon fonctionnement de cet organe est permis, entre autres, par les protéases à sérine qui sont des enzymes dont l'altération peut causer des maladies de la peau. Pour étudier l'importance de la régulation de CAP1/Prss8, une protéase à sérine exprimée au niveau de l'épiderme, des souris génétiquement modifiées, dans lesquelles CAP1/Prss8 est exprimé d'une manière ectopique dans la peau, ont été générées. Les animaux transgéniques pour CAP1/Prss8 présentent une peau squameuse, un épiderme hypertrophique, des processus inflammatoires et se grattent. Ce phénotype a pu être complètement guéri lorsque le gène de PAR2, un récepteur qui règle l'activité des cellules de l'épiderme, est inactivé chez la souris. Ceci montre que PAR2 est une cible de CAP1/Prss8 dans le système étudié. Des études expérimentales suggèrent de plus que l'effet de CAP1/Prss8 dans ce modèle ne dépend pas de son activité enzymatique. En dernière analyse, il a été démontré que l'expression transgénique de nexin-1, un inhibiteur des protéases à sérine exprimé dans la peau, a la capacité d'améliorer la peau squameuse et l'épiderme hypertrophique causés par CAP1/Prss8 transgénique. Les animaux transgéniques pour CAP1/Prss8 et PAR2, et la compréhension du mécanisme d'interaction entre eux, pourraient aider à développer et à tester des molécules inhibitrices de CAP1 /Prss8 et PARI qui pourraient alors être utilisées comme médicaments pour traiter des maladies de la peau comme les ichthyoses.

Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHA0 with enhanced antibiotic production

Introduction of the recombinant cosmid pME3090 into Pseudomonas fluorescens strain CHAO, a good biocontrol agent of various diseases caused by soilborne pathogens, increased three- to five-fold the production of the antibiotic metabolites pyoluteorin (Pit) and 2,4-diacetylphlorogIucinol (Phi) in vitro. Strain CHAO/pME3090 also overproduced Pit and Phi in the rhizosphere of wheat infected or not infected with Pythium ultimum. The biocontrol activity of the wild-type and recombinant Straitis was compared using various plant pathogen-host combinations in a gnotobiotic system. Antibiotic overproduction affected neither the protection of wheat against P. ultimum and Gaeumannomyces graminis var. tritici nor the growth of wheat plants. In contrast, strain CHA0/pME3090 showed an increased capacity to protect cucumber against Fusarium oxysporum f. sp. cucumerinum and Phomopsis sclerotioides, compared with the wild-type strain CHAO, The antibiotic overproducing strain protected tobacco roots significantly better against Thielaviopsis basicola than the wild-type strain but drastically reduced the growth of tobacco plants and was also toxic to the growth of sweet com. On King's B agar and on malt agar, the recombinant strain CHA0/pME3090 inhibited all pathogens more than did the parental strain CHAO. Synthetic Pit and Phi were toxic to all fungi tested. Tobacco and sweet com were more sensitive to synthetic Pit and Phi than were cucumber and wheat. There was no correlation between the sensitivity of the pathogens to the synthetic antibiotics and the degree of disease suppression by strain CHAO pME3090. However, there was a correlation between the sensitivity of the plants and the toxicity of the recombinant strain. We conclude that the plant species rather than the pathogen determines whether cosmid pME3090 in P. fluorescens strain CHAO leads to improved disease suppression.

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.