Identification of a Pseudomonas aeruginosa PAO1 DNA methyltransferase, its Targets, and physiological roles

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N(6)-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria

Stochastic Dispersal Rather Than Deterministic Selection Explains the Spatio-Temporal Distribution of Soil Bacteria in a Temperate Grassland

Spatial and temporal processes shaping microbial communities are inseparably linked but rarely studied together. By Illumina 16S rRNA sequencing, we monitored soil bacteria in 360 stations on a 100 square meter plot distributed across six intra-annual samplings in a rarely managed, temperate grassland. Using a multi-tiered approach, we tested the extent to which stochastic or deterministic processes influenced the composition of local communities. A combination of phylogenetic turnover analysis and null modeling demonstrated that either homogenization by unlimited stochastic dispersal or scenarios, in which neither stochastic processes nor deterministic forces dominated, explained local assembly processes. Thus, the majority of all sampled communities (82%) was rather homogeneous with no significant changes in abundance-weighted composition. However, we detected strong and uniform taxonomic shifts within just nine samples in early summer. Thus, community snapshots sampled from single points in time or space do not necessarily reflect a representative community state. The potential for change despite the overall homogeneity was further demonstrated when the focus shifted to the rare biosphere. Rare OTU turnover, rather than nestedness, characterized abundance-independent β-diversity. Accordingly, boosted generalized additive models encompassing spatial, temporal and environmental variables revealed strong and highly diverse effects of space on OTU abundance, even within the same genus. This pure spatial effect increased with decreasing OTU abundance and frequency, whereas soil moisture – the most important environmental variable – had an opposite effect by impacting abundant OTUs more than the rare ones. These results indicate that – despite considerable oscillation in space and time – the abundant and resident OTUs provide a community backbone that supports much higher β-diversity of a dynamic rare biosphere. Our findings reveal complex interactions among space, time, and environmental filters within bacterial communities in a long-established temperate grassland

Complete Genome Sequence of the Barley Pathogen Xanthomonas translucens pv. translucens DSM 18974 T (ATCC 19319 T)

Jaenicke S, Bunk B, Wibberg D, et al. Complete Genome Sequence of the Barley Pathogen Xanthomonas translucens pv. translucens DSM 18974 T (ATCC 19319 T). Genome Announcements. 2016;4(6): e01334-16.We report here the complete 4.7-Mb genome sequence of Xanthomonas translucens pv. translucens DSM 18974T, which causes black chaff disease on barley (Hordeum vulgare). Genome data of this X. translucens type strain will improve our understanding of this bacterial species

Severe malaria in children leads to a significant impairment of transitory otoacoustic emissions--a prospective multicenter cohort study.

BACKGROUND: Severe malaria may influence inner ear function, although this possibility has not been examined prospectively. In a retrospective analysis, hearing impairment was found in 9 of 23 patients with cerebral malaria. An objective method to quickly evaluate the function of the inner ear are the otoacoustic emissions. Negative transient otoacoustic emissions are associated with a threshold shift of 20 dB and above. METHODS: This prospective multicenter study analyses otoacoustic emissions in patients with severe malaria up to the age of 10 years. In three study sites (Ghana, Gabon, Kenya) 144 patients with severe malaria and 108 control children were included. All malaria patients were treated with parental artesunate. RESULTS: In the control group, 92.6 % (n = 108, 95 % confidence interval 86.19-6.2 %) passed otoacoustic emission screening. In malaria patients, 58.5 % (n = 94, malaria vs controls p < 0.001, 95 % confidence interval 48.4-67.9 %) passed otoacoustic emission screening at the baseline measurement. The value increased to 65.2 % (n = 66, p < 0.001, 95 % confidence interval 53.1-75.5 %) at follow up 14-28 days after diagnosis of malaria. The study population was divided into severe non-cerebral malaria and severe malaria with neurological symptoms (cerebral malaria). Whereas otoacoustic emissions in severe malaria improved to a passing percentage of 72.9 % (n = 48, 95 % confidence interval 59-83.4 %) at follow-up, the patients with cerebral malaria showed a drop in the passing percentage to 33 % (n = 18) 3-7 days after diagnosis. This shows a significant impairment in the cerebral malaria group (p = 0.012 at days 3-7, 95 % confidence interval 16.3-56.3 %; p = 0.031 at day 14-28, 95 % confidence interval 24.5-66.3 %). CONCLUSION: The presented data show that 40 % of children have involvement of the inner ear early in severe malaria. In children, audiological screening after severe malaria infection is not currently recommended, but is worth investigating in larger studies

Genotoxic and cytotoxic effects of snuff on human nasal mucosa cells and lymphocytes

Hintergrund: Die Studienlage zu Kautabak und Zigarettenrauch ist eindeutig und zeigt karzinogenes Potential. Über Schnupftabak ist hingegen wenig bekannt, vor allem auf zellulärer Ebene gibt es keine ausreichenden wissenschaftlichen Publikationen. Somit lässt sich die eventuell mutagene Wirkung von Schnupftabak nur schwer einschätzen. In Konsequenz stützt sich die WHO in ihrer Einstufung des Schnupftabaks als nicht karzinogen auf eine sehr eingeschränkte Datenlage. Ziel: Ziel der vorliegenden Arbeit war es, Schnupftabak auf mögliche zyto- und genotoxische Effekte auf humane Lymphozyten und Nasenschleimhautzellen zu untersuchen um ggf. tumorinitiierende Effekte darzustellen. Material und Methoden: Es kam eine Schnupftabaksorte ohne Menthol und eine Sorte mit Mentholzusatz zum EInsatz. Die benötigten Nasenschleimhautzellen und Lymphozyten wurden von 10 Probanden gewonnen und eine Stunde lang mit einem Schnupftabak-DMSO-Gemisch (2000µg/ml bis 0,01µg/ml) inkubiert. Zur Analyse wurde der Trypanblautest, dee Comet Assay und der Mikrokerntest verwendet. Ergebnis: Der Trypanblautest zeigte keinen Abfall der Vitalität. Beim Comet Assay ergab sich bei Lymphozyten ein signifikanter Anstieg der DNA-Fragmentierung ab 100µg/ml, bei Nasenschleimhautzellen ab 1000µg/ml. Der Mikrokerntest wies keine signifikante Zunahme der Mikrokerne auf. Es konnte kein Unterschied zwischen den beiden Tabaksorten aufgezeigt werden. Diskussion: Es zeigte sich eine Schädigung der Erbsubstanz im Comet Assay, die möglicherweise reparabel ist. Irreparable DNA-Schäden im Sinne von Mikrokernen wurden nicht gefunden. Nach diesen Ergebnissen muss die Einstufung der WHO in Zweifel gezogen werden. Untersuchungen mit weiteren Endpunkten der Genotoxizität sind somit gerechtfertigt, um zu einer fundierten Beurteilung des Risikopotentials von Schnupftabak zu gelangen.Background: While an abundant number of studies concerning tobacco smoke and chewing tobacco show carcinogenic potential, there is little data on the consequences of snuff, especially on the cellular level. Therefore, the mutagenic effect of snuff is hard to estimate and the WHO assessment of snuff being not carcinogenic bases on very limited data. Objectives: This paper investigates potential cytotoxic and genotoxic effects of snuff on human lymphocytes and nasal mucosa cells. Materials and methods: Two kinds of snuff were used, one with a high degree of essential oil. The necessary nasal mucosa cells and lymphocytes were taken from 10 subjects undergoing nasal obstruction surgery and incubated with a snuff mixture (from 0,01µg/ml to 2000µg/ml). Methods included the trypan blue test, the comet assay and the micronucleus test. Results: The trypan blue test showed no decrease in cell viability for both cell types. The comet assay revealed a significant increase in the Olive Tail Moment for lymphocytes starting at 100µg/ml and 1000µg/ml for nasal mucosa cells. There was no significant increase in micronuclei according to the micronucleus test. Conclusion: The present study demonstrated genotoxic damage, such as DNA strand breaks, which may be repaired, but no non-repairable elevated micronuclei. The present findings cast doubts the WHO assessment that snuff is not carcinogenic, however, further research on various genotoxic endpoints in human cells are warranted

Immunantwort bei persistierenden bakteriellen Infektionen: Identifizierung von Borrelia burgdorferi sensu lato und Chlamydia pneumoniae Antigenen

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis (LB), and Chlamydia pneumoniae, a common cause of respiratory tract infections, are able to establish persistent infections that can be harmful to the host. Both pathogens lack an adequate serology, which appears to be linked to unique host immune responses associated with different immune evasion strategies of these bacteria.Beside the clinical manifestations, the diagnosis of Borrelia infections is commonly based on serological testing, which has major shortcomings concerning sensitivity and specificity. The performance of currently available serological tests might be improved by using more sensitive and more specific Borrelia antigens. In the first part of this thesis, novel Borrelia antigens were indentified by phage display technology and characterized with regard to their diagnostic value.- Genomic phage surface display libraries were generated from B. afzelii, B. burgdorferi and B. garinii. Affinity selection against IgG from LB patients revealed nine different Borrelia proteins, including the well established antigen BBK32. Another identified protein, the ribosomal protein L25, was demonstrated to be antigenic by enzyme linked immunosorbent assay using sera from 80 LB patients and 75 controls. The specificity and sensitivity of the antigen was 99% and 23%, respectively, qualifying L25 as a useful antigen when combined with others.The development of a sensitive and specific C. pneumoniae serodiagnosis remains difficult because of the limited knowledge about appropriate antigens. To date, C. pneumoniae serology cannot distinguish between past and persistent infection, which represents a major shortcoming. In the second part of this thesis, novel C. pneumoniae antigens were identified and characterized using immunoproteomics. Further, it was evaluated whether the identified antigens can be used to discriminate past from persistent infections.- Using a proteomic approach combined with immunoblotting, we identified 31 major C. pneumoniae antigens, including 15 novel Chlamydia antigens that showed reactivity towards IgG antibodies. When analyzing the intensity of immunoreactive spots among 19 donors with and 15 donors without evidence for persistent infection, we identified 8 persistence associated antigens and 4 antigens that were associated with non persistent infections. These data represent a basis for a serodiagnosis allowing the identification of persistently infected individuals.In the third part of this thesis, the value of C. pneumoniae induced T cell responses for discriminating persistent from non-persistent infections was investigated.- C. pneumoniae induced T cell responses in different donors were analyzed by flow cytometric multiparameter analysis. After stimulation of PBMC with whole bacteria or cytosol, up to 0.12% activated CD4+ T cells were detected, while no CD8+ T cell activation was found. The characterization of the activated CD4+ T cells with regard to their cytokine production and their stability, argued for memory CD4+ T cells primed during persistent infections. Indeed, C. pneumoniae induced memory CD4+ T cell responses were detected in 9 out of 17 donors with evidence, but only in 1 out of 10 donors without evidence for persistent infections, which is of potential interest for diagnostic applications.Innate immune recognition of peptidoglycan (PGN) has a key role in the onset of antigen specific T cell responses and subsequent antibody production. Thus, infections with Chlamydia, a pathogen that lack detectable amounts of PGN, might provoke suboptimal adaptive immune responses possibly contributing to chlamydial persistence. Since the synthesis of chlamydial PGN is being debated, in the fourth part of the thesis, the transcription of chlamydial genes associated with PGN synthesis was analyzed.Highly sensitive and specific real time PCRs were developed to analyze the transcript levels of nine chlamydial genes associated with PGN synthesis. All genes were strongly upregulated during the C. pneumoniae infection cycle in HEp 2 cells arguing for an active PGN synthesis pathway. The transcription kinetics of these genes closely coincided with the early replication phase of C. pneumoniae indicating a role for the presence of PGN in dividing chlamydial reticulate bodies (RB). Since the transcription was markedly decreased at a time when the RB differentiated back into elementary bodies (EB), PGN is likely not, or only in low amounts, present in Chlamydia EB.In summary, this thesis contributes to the understanding of acquired immune responses against B. burgdorferi sensu lato and C. pneumoniae with regard to diagnostic applications. The presented data provide not only a platform for the development of new diagnostic tools that possibly overcome the current limitations but also might be of interest for vaccination and treatment strategies

Mammalian Display Platform for the Maturation of Bispecific TCR-Based Molecules

Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, but also requires a complex maturation process to increase the naturally low affinity and stability of TCRs. Even though TCR domains can be matured via phage and yeast display, these techniques share the disadvantages of non-human glycosylation patterns and the need for a later reformatting into the final bispecific format. Here, we describe the development and application of a Chinese Hamster Ovary (CHO) display for affinity engineering of TCRs in the context of the final bispecific TCR format. The recombinase-mediated cassette exchange (RCME)-based system allows for stable, single-copy integration of bispecific TCR molecules with high efficiency into a defined genetic locus of CHO cells. We used the system to isolate affinity-increased variants of bispecific T cell engaging receptor (TCER) molecules from a library encoding different CDR variants of a model TCR targeting preferentially expressed antigen in melanoma (PRAME). When expressed as a soluble protein, the selected TCER molecules exhibited strong reactivity against PRAME-positive tumor cells associated with a pronounced cytokine release from activated T cells. The obtained data support the usage of the CHO display-based maturation system for TCR affinity maturation in the context of the final bispecific TCER format

Biomarker candidates of Chlamydophila pneumoniae proteins and protein fragments identified by affinity-proteomics using FTICR-MS and LC-MS/MS

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates

Forward-looking automated cooperative longitudinal control: Extending cooperative adaptive cruise control (CACC) with column-wide reach and automated network quality assessment

Cooperative automated Driver Assistance Systems (CoDAS) are a novel category of advanced driver assistance systems (ADAS), taking into account information received over wireless transmission from other vehicles and roadside infrastructure to enable or improve automated vehicle guidance and control. Research in cooperative longitudinal control so far focusses on improved distance control to a leading cooperative vehicle, often referred to as cooperative adaptive cruise control (CACC). In this work we expand on the concept of cooperative longitudinal control by introducing multi-object control planning, taking into account not only the leading vehicle, but also shared situational knowledge between vehicles and infrastructure. Thus, our system is able to track vehicles directly ahead either from direct cooperation or from transmitted knowledge and can adapt longitudinal control appropriately. We utilize the Collaborative Maneuver Protocol (CMP) to extend vehicle knowledge and estimate network quality. We have evaluated the function in various scenarios in the PHABMACS simulator and discuss effects of transmission quality on control parameters