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Legionella are Gram-negative bacteria that are ubiquitously present in natural and man-made water reservoirs. When humans inhale aerosolized water contaminated with Legionella, alveolar macrophages can be infected, which may lead to a life-threatening pneumonia called Legionnaires' disease. Due to the universal distribution of Legionella in water and their potential threat to human health, the Legionella concentration in water for human use must be strictly monitored, which is difficult since the standard detection still relies on lengthy cultivation and analysis of bacterial morphology. In this study, an antibody against L. pneumophila has been generated from the naïve human HAL antibody libraries by phage-display for the first time. The panning was performed on whole bacterial cells in order to select antibodies that bind specifically to the cell surface of untreated Legionella. The bacterial cell wall component lipopolysaccharide (LPS) was identified as the target structure. Specific binding to the important pathogenic L. pneumophila strains Corby, Philadelphia-1 and Knoxville was observed, while no binding was detected to seven members of the families Enterobacteriaceae, Pseudomonadaceae or Clostridiaceae. Production of this antibody in the recombinant scFv-Fc format using either a murine or a human Fc part allowed the set-up of a sandwich-ELISA for detection of Legionella cells. The scFv-Fc construct proved to be very stable, even when stored for several weeks at elevated temperatures. A sensitivity limit of 4,000 cells was achieved. The scFv-Fc antibody pair was integrated on a biosensor, demonstrating the specific and fast detection of L. pneumophila on a portable device. With this system, 10,000 Legionella cells were detected within 35 min. Combined with a water filtration/concentration system, this antibody may be developed into a promising reagent for rapid on-site Legionella monitoring.

‘You have cancer’ – a devastating diagnosis that still strikes patients hard. Despite substantial improvements of standard therapies over the years, there is still no general cure available. Here, we review the revival of an old concept – the use of bacteria as cancer therapeutics. Bacteria‐mediated tumor therapy has great potential to evolve into a powerful tool against malignant solid tumors.

The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites’ ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.

Cholinergic neuromodulation in the hippocampus controls the salience of background context memory acquired in the presence of elemental stimuli predicting an aversive reinforcement. With pharmacogenetic inhibition we here demonstrate that hilar perforant path-associated (HIPP) cells of the dentate gyrus mediate the devaluation of background context memory during Pavlovian fear conditioning. The salience adjustment is sensitive to reduction of hilar neuropeptide Y (NPY) expression via dominant negative CREB expression in HIPP cells and to acute blockage of NPY-Y1 receptors in the dentate gyrus during conditioning. We show that NPY transmission and HIPP cell activity contribute to inhibitory effects of acetylcholine in the dentate gyrus and that M1 muscarinic receptors mediate the cholinergic activation of HIPP cells as well as their control of background context salience. Our data provide evidence for a peptidergic local circuit in the dentate gyrus that mediates the cholinergic encoding of background context salience during fear memory acquisition., Intra-hippocampal circuits are essential for associating a background context with behaviorally salient stimuli and involve cholinergic modulation at SST+ interneurons. Here the authors show that the salience of the background context memory is modulated through muscarinic activation of NPY+ hilar perforant path associated interneurons and NPY signaling in the dentate gyrus.

promoting vaginal births after caesarean section (VBAC) for eligible women and increasing rates of successful VBACs are the best strategies to reduce the number of repeat caesarean sections (CS). Knowledge of factors that are associated with women's decision-making around mode of birth after CS is important when developing strategies to promote VBAC. This study assessed which factors are associated with women's preferences for VBAC versus elective repeat caesarean section (ERCS) in a new pregnancy after one previous caesarean in Switzerland.cross-sectional web-survey.Western Switzerland.French-speaking women living in Western Switzerland, with one previous CS who gave birth subsequently to a child after a complication-free pregnancy were eligible to participate in the survey. Of 393 women who started the survey in November/December 2014, 349 were included: 227 who planned a VBAC and 122 who planned an ERCS at term.univariable and multivariable analyses were conducted to describe and compare women who had planned a VBAC with women who had planned an ERCS in a pregnancy following a CS. Logistic regression modelling was used to investigate predictors that were associated with a preference for a VBAC at term. Analyses were performed with SPSS 22 and Stata 13.of the women planning a VBAC, 62.6% VBAC gave birth vaginally. Predictors which were significantly associated with increased odds of women choosing a VBAC: duration since previous birth in years (OR=1.11 95% CI [1.03-1.20], p=0.010), having had midwifery care during pregnancy (OR=2.09, 95% CI [1.08-4.05], p=0.029), being advised by their healthcare provider to attempt a VBAC (OR=4.20, 95% CI [1.75-10.09], p=0.001), preference for VBAC during the third trimester of their pregnancy (OR=3.98, 95% CI [1.77-8.93], p=0.001), and wishing to let the child choose the moment of birth (OR=1.46, 95% CI[1.22-1.74], p0.001). The importance of safety for the mother decreased the odds of women preferring a VBAC (OR=0.74, 95% CI [0.60-0.90], p=0.003) while a motivation for more immediate bonding with the baby after birth increased the odds of preferring a VBAC at term (OR=1.25, 95% CI [1.06-1.46], p=0.007).caregivers' recommendations about mode of birth after CS, women's preferences during the third trimester and midwifery care during pregnancy were found to be the most important predictors for preferring a VBAC at term. These results indicate that midwifery antenatal care might be a key factor for fostering women's preference for a VBAC.women with a history of CS who feel ambivalent about the mode of birth are likely to benefit from access to midwifery support.

This study describes the laboratory cultivation of ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganisms). After 2.5 years of successive transfers in an anoxic medium containing ferric sulfate as an electron acceptor, a consortium was attained that is comprised of two members of the order Thermoplasmatales, a member of a proposed ARMAN group, as well as a fungus. The 16S rRNA identity of one archaeon is only 91.6% compared to the most closely related isolate Thermogymnomonas acidicola. Hence, this organism is the first member of a new genus. The enrichment culture is dominated by this microorganism and the ARMAN. The third archaeon in the community seems to be present in minor quantities and has a 100% 16S rRNA identity to the recently isolated Cuniculiplasma divulgatum. The enriched ARMAN species is most probably incapable of sugar metabolism because the key genes for sugar catabolism and anabolism could not be identified in the metagenome. Metatranscriptomic analysis suggests that the TCA cycle funneled with amino acids is the main metabolic pathway used by the archaea of the community. Microscopic analysis revealed that growth of the ARMAN is supported by the formation of cell aggregates. These might enable feeding of the ARMAN by or on other community members.

Human cytomegalovirus (HCMV) encodes numerous proteins and microRNAs that function to evade the immune response and allow the virus to replicate and disseminate in the face of a competent innate and acquired immune system. The establishment of a latent infection by CMV, which if completely quiescent at the level of viral gene expression would represent an ultimate in immune evasion strategies, is not sufficient for lifelong persistence and dissemination of the virus. CMV needs to reactivate and replicate in a lytic cycle of infection in order to disseminate further, which occurs in the face of a fully primed secondary immune response. Without reactivation, latency itself would be redundant for the virus. It is also becoming clear that latency is not a totally quiescent state, but is characterized by limited viral gene expression. Therefore, the virus also needs immune evasion strategies during latency. An effective immune response to CMV is required or viral replication will cause morbidity and ultimately mortality in the host. There is clearly a complex balance between virus immune evasion and host immune recognition over a lifetime. This poses the important question of whether long-term evasion or manipulation of the immune response driven by CMV is detrimental to health. In this meeting report, three groups used the murine model of CMV (MCMV) to examine if the contribution of the virus to immune senescence is set by the (i) initial viral inoculum, (ii) inflation of T cell responses, (iii) or the balance between functionally distinct effector CD4+ T cells. The work of other groups studying the CMV response in humans is discussed. Their work asks whether the ability to make immune responses to new antigens is compromised by (i) age and HCMV carriage, (ii) long-term exposure to HCMV giving rise to an overall immunosuppressive environment and increased levels of latent virus, or (iii) adapted virus mutants (used as potential vaccines) that have the capacity to elicit conventional and unconventional T cell responses.

Background Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. Methods A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples. Results Our results revealed that the generated libraries presented a low average raw error rate per base (95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers. Conclusions The developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-0987-5) contains supplementary material, which is available to authorized users.

Blood culture (BC) sampling rates in Germany are considerably lower than recommended. Aim of our study was to assess knowledge, attitudes, and practice of physicians in Germany regarding BC diagnostics. We conducted a cross-sectional mixed-methods study among physicians working in inpatient care in Germany. Based on the results of qualitative focus groups, a questionnaire-based quantitative study was conducted in 2015–2016. In total, 706 medical doctors and final-year medical students from 11 out of 16 federal states in Germany participated. BC sampling was considered an important diagnostic tool by 95% of the participants. However, only 23% of them would collect BCs in three scenarios for which BC ordering is recommended by present guidelines in Germany; almost one out of ten physicians would not have taken blood cultures in any of the three scenarios. The majority of participants (74%) reported not to adhere to the guideline recommendation that blood culture sampling should include at least two blood culture sets from two different injection sites. High routine in blood culture sampling, perceived importance of blood culture diagnostics, the availability of an in-house microbiological lab, and the department the physician worked in were identified as predictors for good blood culture practice. Our study suggests that there are substantial deficits in BC ordering and the application of guidelines for good BC practice in Germany. Based on these findings, multimodal interventions appear necessary for improving BC diagnostics.

The cell wall of free-living bacteria consists of peptidoglycan (PG) and is critical for maintenance of shape as dissolved solutes cause osmotic pressure and challenge cell integrity. Surprisingly, the subdivision 4 of the phylum Verrucomicrobia appears to be exceptional in this respect. Organisms of this subdivision are described to be devoid of muramic or diaminopimelic acid, usually found as components of PG in bacterial cell walls. Here we describe three novel bacterial strains from a freshwater lake, IG15T, IG16bT and IG31T, belonging to a new genus in the subdivision 4 of Verrucomicrobia which we found to possess PG as part of their cell walls. Biochemical analysis revealed the presence of diaminopimelic acid not only in these novel strains, but also in Opitutus terrae PB90-1T, the closest described relative of strains IG15T, IG16bT and IG31T. Furthermore, we found that nearly all genes necessary for peptidoglycan synthesis are present in genomes of subdivision 4 members, as well as in the complete genome sequence of strain IG16bT. In addition, we isolated and visualized PG-sacculi for strain IG16bT. Thus, our results challenge the concept of peptidoglycan-less free-living bacteria. Our polyphasic taxonomy approach places the novel strains in a new genus within the family Opitutaceae, for which the name Lacunisphaera gen. nov. is proposed. Strain designations for IG15T, IG16bT and IG31T are Lacunisphaera parvula sp. nov.(=DSM 26814 = LMG 29468), Lacunisphaera limnophila sp. nov. (=DSM 26815 = LMG 29469) and Lacunisphaera anatis sp. nov. (=DSM 103142 = LMG 29578) respectively, with Lacunisphaera limnophila IG16bT being the type species of the genus.

Regulatory T (Treg) cells are critical for the shutdown of immune responses and have emerged as valuable targets of immunotherapies. Treg cells can rapidly proliferate; however, the homeostatic processes that limit excessive Treg cell numbers are poorly understood. Here, we show that, compared to conventional T cells, Treg cells have a high apoptosis rate ex vivo correlating with low c-FLIP expression. Treg-specific deletion of c-FLIP in mice resulted in fatal autoimmune disease of a scurfy-like phenotype characterized by absent peripheral Treg cells, activation of effector cells, multi-organ immune cell infiltration, and premature death. Surprisingly, blocking CD95L did not rescue Treg survival in vivo, suggesting additional survival functions of c-FLIP in Treg cells in addition to its classical role in the inhibition of death receptor signaling. Thus, our data reveal a central role for c-FLIP in Treg cell homeostasis and prevention of autoimmunity.

Influenza A virus (IAV) and Streptococcus pneumoniae are major causes of respiratory tract infections, particularly during coinfection. The synergism between these two pathogens is characterized by a complex network of dysregulated immune responses, some of which last until recovery following IAV infection. Despite the high serotype diversity of S. pneumoniae and the serotype replacement observed since the introduction of conjugate vaccines, little is known about pneumococcal strain dependency in the enhanced susceptibility to severe secondary S. pneumoniae infection following IAV infection. Thus, we studied how preinfection with IAV alters host susceptibility to different S. pneumoniae strains with various degrees of invasiveness using a highly invasive serotype 4 strain, an invasive serotype 7F strain, and a carrier serotype 19F strain. A murine model of pneumococcal coinfection during the acute phase of IAV infection showed a significantly increased degree of pneumonia and mortality for all tested pneumococcal strains at otherwise sublethal doses. The incidence and kinetics of systemic dissemination, however, remained bacterial strain dependent. Furthermore, we observed strain-specific alterations in the pulmonary levels of alveolar macrophages, neutrophils, and inflammatory mediators ultimately affecting immunopathology. During the recovery phase following IAV infection, bacterial growth in the lungs and systemic dissemination were enhanced in a strain-dependent manner. Altogether, this study shows that acute IAV infection predisposes the host to lethal S. pneumoniae infection irrespective of the pneumococcal serotype, while the long-lasting synergism between IAV and S. pneumoniae is bacterial strain dependent. These results hold implications for developing tailored therapeutic treatment regimens for dual infections during future IAV outbreaks.

Complex microbial communities inhabit vertebrate digestive systems but thorough understanding of the ecological dynamics and functions of host-associated microbiota within natural habitats is limited. We investigate the role of environmental conditions in shaping gut and skin microbiota under natural conditions by performing a field survey and reciprocal transfer experiments with salamander larvae inhabiting two distinct habitats (ponds and streams). We show that gut and skin microbiota are habitat-specific, demonstrating environmental factors mediate community structure. Reciprocal transfer reveals that gut microbiota, but not skin microbiota, responds differentially to environmental change. Stream-to-pond larvae shift their gut microbiota to that of pond-to-pond larvae, whereas pond-to-stream larvae change to a community structure distinct from both habitat controls. Predicted functions, however, match that of larvae from the destination habitats in both cases. Thus, microbial function can be matched without taxonomic coherence and gut microbiota appears to exhibit metagenomic plasticity., Host-associated microbial communities can shift in structure or function when hosts change locations. Bletz et al. reciprocally transfer salamander larvae between pond and stream habitats to show that gut microbiomes shift in function, but not necessarily taxonomic identities, when hosts encounter a new environment.

The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and Δ tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the Δ tatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo . This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.

Background Reliability of measurements is a prerequisite of medical research. For nominal data, Fleiss’ kappa (in the following labelled as Fleiss’ K) and Krippendorff’s alpha provide the highest flexibility of the available reliability measures with respect to number of raters and categories. Our aim was to investigate which measures and which confidence intervals provide the best statistical properties for the assessment of inter-rater reliability in different situations. Methods We performed a large simulation study to investigate the precision of the estimates for Fleiss’ K and Krippendorff’s alpha and to determine the empirical coverage probability of the corresponding confidence intervals (asymptotic for Fleiss’ K and bootstrap for both measures). Furthermore, we compared measures and confidence intervals in a real world case study. Results Point estimates of Fleiss’ K and Krippendorff’s alpha did not differ from each other in all scenarios. In the case of missing data (completely at random), Krippendorff’s alpha provided stable estimates, while the complete case analysis approach for Fleiss’ K led to biased estimates. For shifted null hypotheses, the coverage probability of the asymptotic confidence interval for Fleiss’ K was low, while the bootstrap confidence intervals for both measures provided a coverage probability close to the theoretical one. Conclusions Fleiss’ K and Krippendorff’s alpha with bootstrap confidence intervals are equally suitable for the analysis of reliability of complete nominal data. The asymptotic confidence interval for Fleiss’ K should not be used. In the case of missing data or data or higher than nominal order, Krippendorff’s alpha is recommended. Together with this article, we provide an R-script for calculating Fleiss’ K and Krippendorff’s alpha and their corresponding bootstrap confidence intervals. Electronic supplementary material The online version of this article (doi:10.1186/s12874-016-0200-9) contains supplementary material, which is available to authorized users.

Hepatocellular carcinoma (HCC) represents a classic example of inflammation-linked cancer. To characterize the role of the immune system in hepatic injury and tumor development, we comparatively studied the extent of liver disease and hepatocarcinogenesis in immunocompromised versus immunocompetent Fah-deficient mice. Strikingly, chronic liver injury and tumor development were markedly suppressed in alymphoid Fah(-/-) mice despite an overall increased mortality. Mechanistically, we show that CD8(+) Tcells and lymphotoxin βare central mediators of HCC formation. Antibody-mediated depletion of CD8(+) Tcells as well as pharmacological inhibition of the lymphotoxin-β receptor markedly delays tumor development in mice with chronic liver injury. Thus, our study unveils distinct functions of the immune system, which are required for liver regeneration, survival, and hepatocarcinogenesis.

Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity.

BackgroundLong noncoding ribonucleic acids (lncRNAs) are a subclass of regulatory noncoding ribonucleic acids for which expression and function in human endothelial cells and angiogenic processes is not well studied.ObjectivesThe authors discovered hypoxia-sensitive human lncRNAs via next-generation ribonucleic acid sequencing and microarray approaches. To address their functional importance in angiogenic processes, several endothelial lncRNAs were characterized for their angiogenic characteristics in vitro and ex vivo.MethodsRibonucleic acid sequencing and microarray-derived data showed specific endothelial lncRNA expression changes after hypoxia. Validation experiments confirmed strong hypoxia-dependent activation of 2 intergenic lncRNAs: LINC00323 and MIR503HG.ResultsSilencing of these lncRNA transcripts led to angiogenic defects, including repression of growth factor signaling and/or the key endothelial transcription factor GATA2. Endothelial loss of these hypoxia-driven lncRNAs impaired cell-cycle control and inhibited capillary formation. The potential clinical importance of these endothelial lncRNAs to vascular structural integrity was demonstrated in an ex vivo model of human induced pluripotent stem cell–based engineered heart tissue.ConclusionsThe authors report an expression atlas of human hypoxia-sensitive lncRNAs and identified 2 lncRNAs with important functions to sustain endothelial cell biology. LncRNAs hold great promise to serve as important future therapeutic targets of cardiovascular disease.

The complement anaphylatoxin C5a contributes to host defense against Staphylococcus aureus. In this study, we investigated the functional role of the two known C5a receptors, C5aR1 and C5aR2, in the host response to S. aureus. We found that C5aR1(-/)(-) mice exhibited greater susceptibility to S. aureus bloodstream infection than wild type and C5aR2(-/)(-) mice, as demonstrated by the significantly higher bacterial loads in the kidneys and heart at 24 h of infection, and by the higher levels of inflammatory IL-6 in serum. Histological and immunohistochemistry investigation of infected kidneys at 24 h after bacterial inoculation revealed a discrete infiltration of neutrophils in wild type mice but already well-developed abscesses consisting of bacterial clusters surrounded by a large number of neutrophils in both C5aR1(-/)(-) and C5aR2(-/)(-) mice. Furthermore, blood neutrophils from C5aR1(-/)(-) mice were less efficient than those from wild type or C5aR2(-/)(-) mice at killing S. aureus. The requirement of C5aR1 for efficient killing of S. aureus was also demonstrated in human blood after disrupting C5a-C5aR1 signaling using specific inhibitors. These results demonstrated a role for C5aR1 in S. aureus clearance as well as a role for both C5aR1 and C5aR2 in the orchestration of the inflammatory response during infection.

Liver infections with hepatotropic viruses, such as hepatitis B virus and hepatitis C virus are accompanied by viral persistence and immune failure. CD8+ T cells are crucial mediators of the intrahepatic antiviral immune response. Chronic infections of the liver and other organs correlate with T-cell exhaustion. It was previously suggested that high antigen load could result in T-cell exhaustion. We aimed at elucidating the impact of different intrahepatic antigen loads on the quality of CD8+ T-cell-mediated immunity by employing an infection-free transgenic mouse model expressing ovalbumin (Ova) as the target antigen. Adoptive transfer of OT-I cells induced a transient intrahepatic immune response toward both high and low Ova levels. However, antigen clearance was achieved only in mice expressing low antigen levels. In contrast, T cells exposed to high antigen levels underwent exhaustion and became depleted, causing antigen persistence. Moreover, when functional T cells were exposed to high intrahepatic antigen levels, a complete transition toward exhaustion was observed. Thus, this study shows that the antigen expression level in the liver correlates inversely with T-cell immunity in vivo and governs the efficiency of immune responses upon antigen presentation.

Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo . Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d 1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa . A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCE Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa . The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium.

Insulin secretion from pancreatic β-cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent-based space-resolved model for SG dynamics in pancreatic β-cells. Using recent experimental data, we consider a single β-cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second-phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the 'newcomer' SGs are recruited from a region within less than 600 nm from the PM.

The Type I interferons (IFN), beta (IFN-β) and the alpha family (IFN-α), act through a common receptor and have anti-inflammatory effects. IFN-β is used to treat multiple sclerosis (MS) and is effective against experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Mice with EAE show elevated levels of Type I IFNs in the central nervous system (CNS), suggesting a role for endogenous Type I IFN during inflammation. However, the therapeutic benefit of Type I IFN produced in the CNS remains to be established. The aim of this study was to examine whether experimentally induced CNS-endogenous Type I IFN influences EAE. Using IFN-β reporter mice, we showed that direct administration of polyinosinic–polycytidylic acid (poly I:C), a potent inducer of IFN-β, into the cerebrospinal fluid induced increased leukocyte numbers and transient upregulation of IFN-β in CD45/CD11b-positive cells located in the meninges and choroid plexus, as well as enhanced IFN-β expression by parenchymal microglial cells. Intrathecal injection of poly I:C to mice showing first symptoms of EAE substantially increased the normal disease-associated expression of IFN-α, IFN-β, interferon regulatory factor-7 and IL-10 in CNS, and disease worsening was prevented for as long as IFN-α/β was expressed. In contrast, there was no therapeutic effect on EAE in poly I:C-treated IFN receptor-deficient mice. IFN-dependent microglial and astrocyte response included production of the chemokine CXCL10. These results show that Type I IFN induced within the CNS can play a protective role in EAE and highlight the role of endogenous type I IFN in mediating neuroprotection. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1418-z) contains supplementary material, which is available to authorized users.

A novel hirsutane-type sesquiterpene with unprecedented modification of the hirsutene scaffold, lentinulactam (1), along with four known metabolites, connatusin A (2), connatusin B (3), 6-hydroxy-2,2-dimethylchroman-4-one (4), and 6-methoxy-2,2-dimethylchroman-4-ol (5) were obtained from the cultures of the basidiomycete fungus Lentinus cf. fasciatus. The absolute configuration of compound 1 was determined on the basis of the NMR spectroscopic data and Mosher ester analysis. The isolation, structure elucidation, and biological evaluation are reported. Lentinulactam (1) is the first hirsutane-type terpenoid containing unusual modification of the hirsutene scaffold represented in 5-6-6 tricyclic skeleton and the bridged lactam ring with an exocyclic double bond. Moreover, lentinulactam (1) represents the first member of hirsutane family having the opposite absolute configuration to those determined for other hirsutanes.

A multigene phylogeny was constructed, including a significant number of representative species of the main lineages in the Xylariaceae and four DNA loci the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, the second largest subunit of the RNA polymerase II (RPB2), and beta-tubulin (TUB2). Specimens were selected based on more than a decade of intensive morphological and chemotaxonomic work, and cautious taxon sampling was performed to cover the major lineages of the Xylariaceae; however, with emphasis on hypoxyloid species. The comprehensive phylogenetic analysis revealed a clear-cut segregation of the Xylariaceae into several major clades, which was well in accordance with previously established morphological and chemotaxonomic concepts. One of these clades contained Annulohypoxylon, Hypoxylon, Daldinia, and other related genera that have stromatal pigments and a nodulisporium-like anamorph. They are accommodated in the family Hypoxylaceae, which is resurrected and emended. Representatives of genera with a nodulisporium-like anamorph and bipartite stromata, lacking stromatal pigments (i.e. Biscogniauxia, Camillea, and Obolarina) appeared in a clade basal to the xylarioid taxa. As they clustered with Graphostroma platystomum, they are accommodated in the Graphostromataceae. The new genus Jackrogersella with J. multiformis as type species is segregated from Annulohypoxylon. The genus Pyrenopolyporus is resurrected for Hypoxylon polyporus and allied species. The genus Daldinia and its allies Entonaema, Rhopalostroma, Ruwenzoria, and Thamnomyces appeared in two separate subclades, which may warrant further splitting of Daldinia in the future, and even Hypoxylon was divided in several clades. However, more species of these genera need to be studied before a conclusive taxonomic rearrangement can be envisaged. Epitypes were designated for several important species in which living cultures and molecular data are available, in order to stabilise the taxonomy of the Xylariales. Fil: Wendt, Lucile. Helmholtz-Zentrum für Infektionsforschung GmbH. Department of Microbial Drugs; Alemania. German Centre for Infection Research; Alemania Fil: Sir, Esteban Benjamin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Unidad Ejecutora Lillo. Fundación Miguel Lillo. Unidad Ejecutora Lillo; Argentina Fil: Kuhnert, Eric. Helmholtz-Zentrum für Infektionsforschung GmbH. Department of Microbial Drugs; Alemania. German Centre for Infection Research; Alemania Fil: Heitkämper, Simone. Helmholtz-Zentrum für Infektionsforschung GmbH. Department of Microbial Drugs; Alemania. German Centre for Infection Research; Alemania Fil: Lambert, Christopher. Helmholtz-Zentrum für Infektionsforschung GmbH. Department of Microbial Drugs; Alemania. German Centre for Infection Research; Alemania Fil: Hladki, Adriana I.. Fundación Miguel Lillo. Dirección de Botánica. Instituto de Micologia; Argentina Fil: Romero, Andrea Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Luangsa-Ard, Janet Jennifer. National Center for Genetic Engineering and Biotechnology; Tailandia Fil: Srikitikulchai, Prasert. National Center for Genetic Engineering and Biotechnology; Tailandia Fil: Peršoh, Derek. Ruhr-Universität Bochum; Alemania Fil: Stadler, Marc. Helmholtz-Zentrum für Infektionsforschung GmbH. Department of Microbial Drugs; Alemania. German Centre for Infection Research; Alemania

The ubiquitously expressed transcriptional regulator serum response factor (SRF) is controlled by both Ras/MAPK (mitogen-activated protein kinase) and Rho/actin signaling pathways, which are frequently activated in hepatocellular carcinoma (HCC). We generated SRF-VP16iHep mice, which conditionally express constitutively active SRF-VP16 in hepatocytes, thereby controlling subsets of both Ras/MAPK- and Rho/actin-stimulated target genes. All SRF-VP16iHep mice develop hyperproliferative liver nodules that progresses to lethal HCC. Some murine (m)HCCs acquire Ctnnb1 mutations equivalent to those in human (h)HCC. The resulting transcript signatures mirror those of a distinct subgroup of hHCCs, with shared activation of oncofetal genes including Igf2, correlating with CpG hypomethylation at the imprinted Igf2/H19 locus. Conclusion: SRF-VP16iHep mHCC reveal convergent Ras/MAPK and Rho/actin signaling as a highly oncogenic driver mechanism for hepatocarcinogenesis. This suggests simultaneous inhibition of Ras/MAPK and Rho/actin signaling as a treatment strategy in hHCC therapy. (Hepatology 2015;61:979–989)

Activated naive CD4+ T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4+ T cells, Th1 and Th17 cells. We could demonstrate that naive CD4+ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.

This deposit is composed by a publication in which the IGC' authors have had the role of collaboration (it's a collaboration publication). This type of deposit in ARCA is in restrictedAccess (it can't be in open access to the public), and could only be accessed by two ways: either by requesting a legal copy to the author (the email contact present in this deposit) or by visiting the following link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383977/ The laboratory mouse is a key model organism to investigate mechanism and therapeutics of human disease. The number of targeted genetic mouse models of disease is growing rapidly due to high-throughput production strategies employed by the International Mouse Phenotyping Consortium (IMPC) and the development of new, more efficient genome engineering techniques such as CRISPR based systems. We have previously described the European Mouse Mutant Archive (EMMA) resource and how this international infrastructure provides archiving and distribution worldwide for mutant mouse strains. EMMA has since evolved into INFRAFRONTIER (http://www.infrafrontier.eu), the pan-European research infrastructure for the systemic phenotyping, archiving and distribution of mouse disease models. Here we describe new features including improved search for mouse strains, support for new embryonic stem cell resources, access to training materials via a comprehensive knowledgebase and the promotion of innovative analytical and diagnostic techniques. European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI).

Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasia.

Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery., Author summary Many bacteria use the bacterial flagellum for directed movement in various environments. The assembly and function of the bacterial flagellum and the related virulence-associated injectisome relies on protein export via a conserved type III secretion system (T3SS). The multicomponent transmembrane core export apparatus of the flagellar T3SS consists of FlhA/B and FliP/Q/R and must assemble in a highly coordinated manner. In the present study, we determined the role of the transmembrane protein FliO in the maturation of the flagellar core protein export apparatus. We show that FliO functions as a flagellum-specific chaperone during the initial step of export apparatus assembly. FliO facilitates the efficient formation of a stable FliP–FliR core complex and is thus required for quality management and productive assembly of the flagellar export apparatus. Our results suggest a coordinated assembly process of the flagellar core export apparatus that nucleates with the FliO-dependent formation of a FliP–FliR complex. Subsequent incorporation of FliQ, FlhB, and FlhA leads to the assembly of a secretion-competent flagellar T3SS.

Soraphen A is a myxobacterial metabolite that blocks the acetyl-coenzyme A carboxylase of the host and was previously identified as a novel HIV inhibitor. Here, we report that soraphen A acts by reducing virus production and altering the gp120 virion content, impacting entry capacity and infectivity. These effects are partially reversed by addition of palmitic acid, suggesting that inhibition of HIV envelope palmitoylation is one of the mechanisms of antiviral action. The research was funded by the German Centre for Infection Research (DZIF), Spanish Ministry of Economy, Industry and Competitiveness and FEDER BES-2011-048569, Spanish Ministry of Economy, Industry and Competitiveness and FEDER SAF2013-46077-R SAF2016-75505-R, the Spanish Ministry of Economy, Industry and Competitiveness and FEDER BFU2016-80039-R and the Maria de Maetzu Programme for Units of Excellence in R&DMDM-2014-0370

Plasmid mediated horizontal gene transfer (HGT) has been speculated to be one of the prime mechanisms for the adaptation of roseobacters (Rhodobacteraceae) to their ecological niches in the marine habitat. Their plasmids contain ecologically crucial functional modules of up to ∼40-kb in size, e.g., for aerobic anoxygenic photosynthesis, flagellar formation and the biosynthesis of the antibiotic tropodithietic acid. Furthermore, the widely present type four secretion system (T4SS) of roseobacters has been shown to mediate conjugation across genus barriers, albeit in the laboratory. Here we discovered that Confluentimicrobium naphthalenivorans NS6(T), a tidal flat bacterium isolated in Korea, carries a 185-kb plasmid, which exhibits a long-range synteny with the conjugative 126-kb plasmid of Dinoroseobacter shibae DFL12(T). Both replicons are stably maintained by RepABC operons of the same compatibility group (-2) and they harbor a homologous T4SS. Principal component analysis of the codon usage shows a large similarity between the two plasmids, while the chromosomes are very distinct, showing that neither of the two bacterial species represents the original host of those RepABC-2 type plasmids. The two species do not share a common habitat today and they are phylogenetically only distantly related. Our finding demonstrates the first clear-cut evidence for conjugational plasmid transfer across biogeographical and phylogenetic barriers in Rhodobacteraceae and documents the importance of conjugative HGT in the ocean.

Periodontitis is an extremely prevalent disease worldwide and is driven by complex dysbiotic microbiota. Here we analyzed the transcriptional activity of the periodontal pocket microbiota from all domains of life as well as the human host in health and chronic periodontitis. Bacteria showed strong enrichment of 18 KEGG functional modules in chronic periodontitis, including bacterial chemotaxis, flagellar assembly, type III secretion system, type III CRISPR-Cas system, and two component system proteins. Upregulation of these functions was driven by the red-complex pathogens and candidate pathogens, e.g. Filifactor alocis, Prevotella intermedia, Fretibacterium fastidiosum and Selenomonas sputigena. Nine virulence factors were strongly up-regulated, among them the arginine deiminase arcA from Porphyromonas gingivalis and Mycoplasma arginini. Viruses and archaea accounted for about 0.1% and 0.22% of total putative mRNA reads, respectively, and a protozoan, Entamoeba gingivalis, was highly enriched in periodontitis. Fourteen human transcripts were enriched in periodontitis, including a gene for a ferric iron binding protein, indicating competition with the microbiota for iron, and genes associated with cancer, namely nucleolar phosphoprotein B23, ankyrin-repeat domain 30B-like protein and beta-enolase. The data provide evidence on the level of gene expression in vivo for the potentially severe impact of the dysbiotic microbiota on human health.

Inflammasomes play a central role in regulating intestinal barrier function and immunity during steady state and disease. Because the discoveries of a passenger mutation and a colitogenic microbiota in the widely used caspase-1-deficient mouse strain have cast doubt on previously identified direct functions of caspase-1, we reassessed the role of caspase-1 in the intestine. To this end, we generated Casp1(-/-) and Casp11(-/-) mice and rederived them into an enhanced barrier facility to standardize the microbiota. We found that caspase-11 does not influence caspase-1-dependent processing of IL-18 in homeostasis and during DSS colitis. Deficiency of caspase-1, but not caspase-11, ameliorated the severity of DSS colitis independent of microbiota composition. Ablation of caspase-1 in intestinal epithelial cells was sufficient to protect mice against DSS colitis. Moreover, Casp1(-/-) mice developed fewer inflammation-induced intestinal tumors than control mice. These data show that canonical inflammasome activation controls caspase-1 activity, contributing to exacerbation of chemical-induced colitis.

// Dino Kocijancic 1, * , Sebastian Felgner 1, 2, * , Tim Schauer 1 , Michael Frahm 1 , Ulrike Heise 3 , Kurt Zimmermann 4 , Marc Erhardt 2 and Siegfried Weiss 1, 5 1 Molecular Immunology, Helmholtz Center for Infection Research, Braunschweig, Germany 2 Infection Biology of Salmonella, Helmholtz Center for Infection Research, Braunschweig, Germany 3 Mouse-Pathology Service Unit, Helmholtz Center for Infection Research, Braunschweig, Germany 4 Symbio Gruppe GmbH & Co KG, Herborn, Germany 5 Institute of Immunology, Medical School Hannover, Hannover, Germany * These authors have contributed equally to this work Correspondence to: Dino Kocijancic, email: dino.kocijancic@helmholtz-hzi.de Keywords: Intra-tumoral injection, Salmonella, E. coli, bacteria mediated tumor therapy, murine tumor model Received: October 29, 2016 Accepted: May 05, 2017 Published: June 07, 2017 ABSTRACT Cancer is a devastating disease and a large socio-economic burden. Novel therapeutic solutions are on the rise, although a cure remains elusive. Application of microorganisms represents an ancient therapeutic strategy, lately revoked and refined via simultaneous attenuation and amelioration of pathogenic properties. Salmonella Typhimurium has prevailed in preclinical development. Yet, using virulent strains for systemic treatment might cause severe side effects. In the present study, we highlight a modified strain based on Salmonella Typhimurium UK-1 expressing hexa-acylated Lipid A. We corroborate improved anti-tumor properties of this strain and investigate to which extent an intra-tumoral (i.t.) route of infection could help improve safety and retain advantages of systemic intravenous (i.v.) application. Our results show that i.t. infection exhibits therapeutic efficacy against CT26 and F1.A11 tumors similar to a systemic route of inoculation. Moreover, i.t. application allows extensive dose titration without compromising tumor colonization. Adverse colonization of healthy organs was generally reduced via i.t. infection and accompanied by less body weight loss of the murine host. Despite local application, adjuvanticity remained, and a CT26-specific CD8 + T cell response was effectively stimulated. Most interestingly, also secondary tumors could be targeted with this strategy, thereby extending the unique tumor targeting ability of Salmonella . The i.t. route of inoculation may reap the benefits of systemic infection and aid in safety assurance while directing potency of an oncolytic vector to where it is most needed, namely the primary tumor.

Five new compounds including the glycosylated β-naphthol (1, akanthol), a glycosylated pyrazine (2, akanthozine), and three amide derivatives including a hydroxamic acid derivative (3–5) were isolated from the spider-associated fungus Akanthomyces novoguineensis (Cordycipitaceae, Ascomycota). Their structures were elucidated by using high resolution mass spectrometry (HRMS) and NMR spectroscopy. In this study, the antimicrobial, cytotoxic, anti-biofilm, and nematicidal activities of the new compounds were evaluated. The distribution pattern of secondary metabolites in the species was also revealed in which more isolates of A. novoguineensis were encountered and their secondary metabolite profiles were examined using analytical HPLC with diode array and mass spectrometric detection (HPLC-DAD/MS). Remarkably, all isolated compounds are specifically produced by A. novoguineensis.

Penicillium sp. F37 has been isolated from the marine sponge Axinella corrugata and shown to be closely related to Penicillium maximae. From the culture of Penicillium sp. F37 arvoredol, a novel chlorinated polyketide with 6,7-dihydro-4(5H)-benzofuranone moiety has been isolated and characterized by spectroscopic methods Arvoredol prevented biofilm formation of the human pathogen Staphylococcus epidermidis at a concentration of 125 μg mL−1 by 40%. It was also active against colorectal carcinoma HCT116 cells with a MIC of 7.9 μg mL−1. © 2017 Phytochemical Society of Europe

Biomaterial-associated Pseudomonas aeruginosa biofilm infections constitute cascade of host immune reactions ultimately leading towards implant failure. Due to lack of relevant in vivo biofilm models, majority of the studies report host immune responses against free living or planktonic bacteria while bacteria in clinical situations live more frequently as biofilm communities than as single cells. Present study investigated host immune responses against biomaterial-associated P. aeruginosa biofilms in a clinically relevant mouse model. Previously, we reported metallic magnesium, a prospective biodegradable implant, to be permissive for bacterial biofilms in vivo even though it exhibits antibacterial properties in vitro. Therefore, magnesium was employed as biomaterial to investigate in vivo biofilm formation and associated host immune responses by using two P. aeruginosa strains and two mouse strains. P. aeruginosa formed biofilms on subcutaneously implanted magnesium discs. Non-invasive in vivo imaging indicated transient inflammatory responses at control sites whereas robust prolonged interferon-β (IFN-β) expression was observed from biofilms in a transgenic animal reporter. Further, immunohistology and electron microscopic results showed that bacterial biofilms were located in two dimensions immediately on the implant surface and at a short distance in the adjacent tissue. These biofilms were surrounded by inflammatory cells (mainly polymorphonuclear cells) as compared to controls. Interestingly, even though the number of live bacteria in various organs remained below detectable levels, splenomegaly indicated systemic inflammatory processes. Overall, these findings confirmed the resistance of biofilm infections in vivo to potentially antibacterial properties of magnesium degradation products. In vivo imaging and histology indicated the induction of both, local and systemic host inflammatory responses against P. aeruginosa biofilms. Even though the innate host immune defenses could not eliminate the local infection for up to two weeks, there was no apparent systemic bacteremia and all animals investigated survived the infection.

// Dino Kocijancic 1 , Sara Leschner 1 , Sebastian Felgner 1 , Ronja-Melinda Komoll 1 , Michael Frahm 1 , Vinay Pawar 1 , Siegfried Weiss 1, 2 1 Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Institute of Immunology, Medical School Hannover, Hannover, Germany Correspondence to: Dino Kocijancic, email: dino.kocijancic@helmholtz-hzi.de Keywords: TNF alpha, LPS, cancer immune therapy, salmonella, bacteria mediated tumor therapy Received: June 15, 2016 Accepted: March 29, 2017 Published: April 06, 2017 ABSTRACT The potential of bacteria-mediated tumor therapy (BMTT) is highlighted by more than a century of investigation. Attenuated Salmonella has prevailed as promising therapeutic agents. For BMTT - categorized as an immune therapy - the exact contribution of particular immune reactions to the therapeutic effect remains ambiguous. In addition, one could argue for or against the requirement of bacterial viability and tumor targeting. Herein we evaluate the isolated therapeutic efficacy of purified LPS and TNF-α, which together account for a dominant immunogenic pathway of gram negative bacteria like Salmonella . We show that therapeutic efficacy against CT26 tumors does not require bacterial viability. Analogous to viable Salmonella SL7207, tumor regression by a specific CD8 + T cell response can be induced by purified LPS or recombinant TNF-α (rTNF-α). Conversely, therapeutic effects against RenCa tumors were abrogated upon bacterial avitalization and limited using isolated adjuvants. This argues for an alternative mechanistic explanation for SL7207 against RenCa that depends on viability and persistence. Unable to boost bacterial therapies by co-injection of rTNF-α suggested therapeutic effects along this axis are exhausted by the intrinsic adjuvanticity of bacteria alone. However, the importance of TNF-α for BMTT was highlighted by its support of tumor invasion and colonization in concert with lower infective doses of Salmonella . In consideration, bacterial therapeutic effectiveness along the axis of LPS and TNF-α appears limited, and does not offer the necessary plasticity for different tumors. This emphasizes a need for recombinant strengthening and vehicular exploitation to accommodate potency, plasticity and distinctiveness in BMTT.

Trapping RNA polymerase in the act The enzyme RNA polymerase (RNAP) finds promoter elements in the genome, separates (or “melts”) the DNA strands, and transcribes the template DNA strand to give RNA. A mobile clamp in RNAP plays a key role in initiating transcription. Feklistov et al. locked the clamp of bacterial RNAP in distinct conformations by using small molecules. They then used fluorescent probes to monitor binding as the promoter DNA was separated. Unexpectedly, they found that the clamp transiently closed to nucleate DNA melting, opened to load single-stranded DNA into the active site, and then closed around the template strand to start transcription. Science , this issue p. 863

During recent years, the analysis of the human microbiota has been receiving more and more scientific focus. Deep sequencing analysis enables characterization of microbial communities in different environments without the need of culture-based methods. Hereby, information about microbial communities is increasing enormously. Numerous studies in humans and animal models revealed the important role of the microbiome in emergence and natural course of diseases such as autoimmune diseases and metabolic disorders, e. g., the metabolic syndrome. The identification of causalities between the intestinal microbiota composition and function, and diseases in humans and animal models can help to develop individualized therapies targeting the microbiome and its modification. Nowadays, it is established that several factors influence the composition of the microbiota. Diet it is one of the major factors shaping the microbiota and the use of pro- and prebiotica may induce changes in the microbial community. Fecal microbiome transfer is the first approach targeting the intestinal microbiota which is implemented in the clinical routine for patients with therapy-refractory infections with Clostridium difficile. Herewith, the recipient's microbiota can be changed permanently and the patient can be cured from the infection.

Background Trimethylamine (TMA), produced by the gut microbiota from dietary quaternary amines (mainly choline and carnitine), is associated with atherosclerosis and severe cardiovascular disease. Currently, little information on the composition of TMA producers in the gut is available due to their low abundance and the requirement of specific functional-based detection methods as many taxa show disparate abilities to produce that compound. Results In order to examine the TMA-forming potential of microbial communities, we established databases for the key genes of the main TMA-synthesis pathways, encoding choline TMA-lyase (cutC) and carnitine oxygenase (cntA), using a multi-level screening approach on 67,134 genomes revealing 1107 and 6738 candidates to exhibit cutC and cntA, respectively. Gene-targeted assays enumerating the TMA-producing community by quantitative PCR and characterizing its composition via Illumina sequencing were developed and applied on human fecal samples (n = 50) where all samples contained potential TMA producers (cutC was detected in all individuals, whereas only 26% harbored cntA) constituting, however, only a minor part of the total community (below 1% in most samples). Obtained cutC amplicons were associated with various taxa, in particular with Clostridium XIVa strains and Eubacterium sp. strain AB3007, though a bulk of sequences displayed low nucleotide identities to references (average 86% ± 7%) indicating that key human TMA producers are yet to be isolated. Co-occurrence analysis revealed specific groups governing the community structure of cutC-exhibiting taxa across samples. CntA amplicons displayed high identities (~99%) to Gammaproteobacteria-derived references, primarily from Escherichia coli. Metagenomic analysis of samples provided by the Human Microbiome Project (n = 154) confirmed the abundance patterns as well as overall taxonomic compositions obtained with our assays, though at much lower resolution, whereas 16S ribosomal RNA gene sequence analysis could not adequately uncover the TMA-producing potential. Conclusions In this study, we developed a diagnostic framework that enabled the quantification and comprehensive characterization of the TMA-producing potential in human fecal samples. The key players were identified, and together with predictions on their environmental niches using functional genomics on most closely related reference strains, we provide crucial information for the development of specific treatment strategies to restrain TMA producers and limit their proliferation. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0271-9) contains supplementary material, which is available to authorized users.

Background Acute respiratory infections (ARI) and acute gastrointestinal infections (AGI) are the most common childhood infections, and corresponding data can either be collected prospectively or retrospectively. The aim of this study was to estimate the incidence of respiratory and gastrointestinal episodes in German households with children attending day care and to compare results of prospective and retrospective data collection. Methods We conducted a 4 months prospective cohort study in the winter period 2014/2015 and recruited parents of children aged 0–6 years in 75 day care centers in Braunschweig, Lower Saxony, Germany. For all household members, we collected information on episodes of ARI and AGI. We applied prospective data collection in one study arm and retrospective data collection with a reporting period of 2 months in the other. Poisson regression was used to model monthly incidence rates for both study arms. Results In total, 100 households (including 404 persons) participated in the retrospective group and 77 households (282 persons) in the prospective group. Incidence estimates for ARI (retrospective group: 0.52 per person month, prospective group: 0.47) were higher than for AGI (retrospective group: 0.14, prospective group: 0.13). The adjusted incidence estimates were similar in both study arms for ARI (incidence rate ratio for retrospective versus prospective data collection: 1.11 [confidence interval (CI) 95% 0.99; 1.24], p = 0.42) as well as for AGI (1.10 [CI 95% 0.89; 1.37], p = 0.27). Conclusion If there is no need to collect biomaterials or data on severity of the diseases, incidence of infections in the household setting over a short time period (2 months) can be assessed retrospectively. Electronic supplementary material The online version of this article (doi:10.1186/s12879-017-2434-5) contains supplementary material, which is available to authorized users.

Streptococcus dysgalactiae subsp. equisimilis belongs to the β-hemolytic group C and G pyogenic group of streptococci. Here, we report the draft genome of the S. dysgalactiae subsp. equisimilis strain C161L1 from Vellore, a region in southern India with a high incidence rate of S. dysgalactiae subsp. equisimilis infection. This genome is 2.1 Mb long, with a 39.82% G+C content, and encodes 2,022 genes.

// Christoph Thurm 1 , Mateusz P. Poltorak 1,5 , Elisa Reimer 2 , Melanie M. Brinkmann 2 , Lars Leichert 3 , Burkhart Schraven 1,4 and Luca Simeoni 1 1 Institute of Molecular and Clinical Immunology, Health Campus Immunology, Infectiology, and Inflammation, Otto von Guericke University, Magdeburg, Germany 2 Viral Immune Modulation Group, Helmholtz Centre for Infection Research, Braunschweig, Germany 3 Institute for Biochemistry and Pathobiochemistry, Ruhr University, Bochum, Germany 4 Department of Immune Control, Helmholtz Centre for Infection Research, Braunschweig, Germany 5 Current address: Institut fur Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universitat Munchen, Munich, Germany Correspondence to: Luca Simeoni, email: // Keywords : Zap70, oxidation, TCR signaling, Cdc37, protein stability, Immunology and Microbiology Section, Immune response, Immunity Received : January 12, 2017 Accepted : March 16, 2017 Published : March 22, 2017 Abstract ζ-associated protein of 70 kDa ( Zap70 ) is crucial for T-cell receptor (TCR) signaling. Loss of Zap70 in both humans and mice results in severe immunodeficiency. On the other hand, the expression of Zap70 in B-cell malignancies correlates with the severity of the disease. Because of its role in immune-related disorders, Zap70 has become a therapeutic target for the treatment of human diseases. It is well-established that the activity/expression of Zap70 is regulated by post-translational modifications of crucial amino acids including the phosphorylation of tyrosines and the ubiquitination of lysines. Here, we have investigated whether also oxidation of cysteine residues regulates Zap70 functions. We have identified C575 as a major sulfenylation site of Zap70. A C575A substitution results in protein instability, reduced activity, and increased dependency on the Hsp90/Cdc37 chaperone system. Indeed, Cdc37 overexpression reconstituted partially the expression but fully the function of Zap70C575A. C575 lies within a Mx (2) CWx (6) R motif which is highly conserved among almost all human tyrosine kinases. Mutation of any of the conserved amino acids, but not of a non-conserved residue preceding the cysteine, also results in Zap70 instability. Collectively, we have identified a new redox-active motif which is crucial for the regulation of Zap70 stability/activity. We believe that this motif has the potential to become a novel target for the development of therapeutic tools to modulate the expression/activity of kinases.

The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia call ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.

The emergence of antibiotic resistance mechanisms among bacterial pathogens increases the demand for novel treatment strategies. Lately, the contribution of non-coding RNAs to antibiotic resistance and their potential value as drug targets became evident. RNA attenuator elements in mRNA leader regions couple expression of resistance genes to the presence of the cognate antibiotic. Trans-encoded small RNAs (sRNAs) modulate antibiotic tolerance by base-pairing with mRNAs encoding functions important for resistance such as metabolic enzymes, drug efflux pumps, or transport proteins. Bacteria respond with extensive changes of their sRNA repertoire to antibiotics. Each antibiotic generates a unique sRNA profile possibly causing downstream effects that may help to overcome the antibiotic challenge. In consequence, regulatory RNAs including sRNAs and their protein interaction partners such as Hfq may prove useful as targets for antimicrobial chemotherapy. Indeed, several compounds have been developed that kill bacteria by mimicking ligands for riboswitches controlling essential genes, demonstrating that regulatory RNA elements are druggable targets. Drugs acting on sRNAs are considered for combined therapies to treat infections. In this review, we address how regulatory RNAs respond to and establish resistance to antibiotics in bacteria. Approaches to target RNAs involved in intrinsic antibiotic resistance or virulence for chemotherapy will be discussed.

Introduction Primary immunodeficiency disorders (PIDs) are a heterogeneous group of more than 200 rare diseases. Timely diagnosis is of uttermost importance. Therefore, we aimed to develop a diagnostic questionnaire with computerized pattern-recognition in order to support physicians to identify suspicious patient histories. Materials and methods Standardized interviews were conducted with guardians of children with PID. The questionnaire based on parental observations was developed using Colaizzis’ framework for content analysis. Answers from 64 PID patients and 62 controls were analyzed by data mining methods in order to make a diagnostic prediction. Performance was evaluated by k-fold stratified cross-validation. Results The diagnostic support tool achieved a diagnostic sensitivity of up to 98%. The analysis of 12 interviews revealed 26 main phenomena observed by parents in the pre-diagnostic period. The questions were systematically phrased and selected resulting in a 36-item questionnaire. This was answered by 126 patients with or without PID to evaluate prediction. Item analysis revealed significant questions. Discussion Our approach proved suitable for recognizing patterns and thus differentiates between observations of PID patients and control groups. These findings provide the basis for developing a tool supporting physicians to consider a PID with a questionnaire. These data support the notion that patient’s experience is a cornerstone in the diagnostic process.