Isolierung und kultivierungsunabhängige Untersuchungen von magnetotaktischen Bakterien aus marinen und limnischen Sedimenten

In this work the diversity and distribution of magnetotactic bacteria (MTB) in Northern Germany were investigated. With the exception of extreme eutrophic environment different MTB (Spirilla, cocci, rods, vibrios) were found in all samples from marine and freshwater environments. Magnetotactic multicellular aggregates were found for the first time in the German Bight and in the Baltic Sea.During the incubation in microcosms an increasing number and a decreasing diversity of MTB was observed. In most cases the MTB population was dominated by magnetotactic cocci and 16S rDNA analysis identified them as Alphaproteobacteria . Up to 11% sequence divergences were found between the MTB of different microcosms but a high variability was also observed within single microcosms at different times.In one microcosm from a lake in Bremen a magnetotactic rod (MHB-1) was enriched which is closely related to Magnetobacterium bavaricum , the only known MTB from the Nitrospira phylum.A correlation between the development of a distinct MTB population in the microcosms and the original geographical location of the samples was not observed but the heterogeneous vertical distribution of MTB indicates an adaption to special gradients. Most MTB (up to 98%) were restricted to anoxic sediment layers and reached up to 1,5 x 107 MTB/cm3 with an abundance of up to 1% of the total cell counts which indicates a significant influence of MTB on the microbial iron cycle.The high effective enrichment of MTB by magnetic collection, race-track , resp. were demonstrated by microscopy, Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA fragments and Amplified Ribosomal DNA Restriction Analysis (ARDRA). The selective enrichment of MTB allowed the construction of a genomic library which most likely contains a fragment of the mamAB cluster.From the several cultivation experiments 10 new magnetotactic spirilla were isolated. All strains are microaerohpilic and members of the genus Magnetospirillum

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Isolation and cultivation independent investigations of magnetotactic bacteria from marine and freshwater sediments

In this work the diversity and distribution of magnetotactic bacteria (MTB) in Northern Germany were investigated. With the exception of extreme eutrophic environment different MTB (Spirilla, cocci, rods, vibrios) were found in all samples from marine and freshwater environments. Magnetotactic multicellular aggregates were found for the first time in the German Bight and in the Baltic Sea.During the incubation in microcosms an increasing number and a decreasing diversity of MTB was observed. In most cases the MTB population was dominated by magnetotactic cocci and 16S rDNA analysis identified them as Alphaproteobacteria . Up to 11% sequence divergences were found between the MTB of different microcosms but a high variability was also observed within single microcosms at different times.In one microcosm from a lake in Bremen a magnetotactic rod (MHB-1) was enriched which is closely related to Magnetobacterium bavaricum , the only known MTB from the Nitrospira phylum.A correlation between the development of a distinct MTB population in the microcosms and the original geographical location of the samples was not observed but the heterogeneous vertical distribution of MTB indicates an adaption to special gradients. Most MTB (up to 98%) were restricted to anoxic sediment layers and reached up to 1,5 x 107 MTB/cm3 with an abundance of up to 1% of the total cell counts which indicates a significant influence of MTB on the microbial iron cycle.The high effective enrichment of MTB by magnetic collection, race-track , resp. were demonstrated by microscopy, Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA fragments and Amplified Ribosomal DNA Restriction Analysis (ARDRA). The selective enrichment of MTB allowed the construction of a genomic library which most likely contains a fragment of the mamAB cluster.From the several cultivation experiments 10 new magnetotactic spirilla were isolated. All strains are microaerohpilic and members of the genus Magnetospirillum

Combined Approach for Characterization of Uncultivated Magnetotactic Bacteria from Various Aquatic Environments

Both magnetic collection and “race track” purification techniques were highly effective for selective enrichment of magnetotactic bacteria (MTB) from complex communities, as suggested by amplified ribosomal DNA restriction analysis and denaturing gradient gel electrophoresis combined with sequence analysis of 16S rRNA genes. Using these purification methods, the occurrence and diversity of MTB in microcosms from various marine and freshwater environments were assayed by using a combined microscopic, molecular, and cultivation approach. Most microcosms were dominated by magnetotactic cocci. Consistently, the majority of retrieved 16S RNA sequences were affiliated with a distinct cluster in the Alphaproteobacteria. Within this lineage the levels of sequence divergence were <1 to 11%, indicating genus-level diversity between magnetotactic cocci from various microcosms, as well as between MTB from different stages of succession of the same microcosms. The community composition in microscosms underwent drastic succession during incubation, and significant heterogeneities were observed between microcosms from the same environmental sources. A novel magnetotactic rod (MHB-1) was detected in a sediment sample from a lake in northern Germany by fluorescence in situ hybridization. MHB-1 falls into the Nitrospira phylum, displaying 91% 16S rRNA sequence similarity to “Magnetobacterium bavaricum.” In extensive cultivation attempts, we failed to isolate MHB-1, as well as most other MTB present in our samples. However, although magnetotactic spirilla were not frequently observed in the enrichments, 10 novel isolates of the genus Magnetospirillum which had not routinely been isolated in pure culture before were obtained

Properties of Intracellular Magnetite Crystals Produced by \u3ci\u3eDesulfovibrio magneticus\u3c/i\u3e Strain RS-1

Desulfovibrio magneticus strain RS-1 is an anaerobic sulfate-reducing bacterium. Cells form intracellular nanocrystals of magnetite but are only weakly magnetotactic. In order to understand the unusual magnetic response of this strain, we studied magnetite crystals within cells grown with fumarate and sulfate. Many cells grown under either condition did not form magnetic crystals while others contained only 1 to 18 small (~40 nm) magnetite-containing magnetosomes. Bulk magnetic measurements of whole cells showed a superparamagnetic-like behavior, indicating that many of the magnetite crystals are too small to have a permanent magnetic moment at ambient temperature. The temperature of the Verwey transition is lower (~86 K) than of magnetite from other magnetotactic strains, likely indicating partial oxidation of magnetite into maghemite. As a result of the small size and small number of magnetite magnetosomes, the magnetic moments of most cells grown anaerobically with fumarate or sulfate are insufficient for magnetotaxis. In addition to intracellular magnetite, in some cultures another iron oxide, hematite, formed on the surfaces of cells. The hematite grains are embedded in an extracellular polymeric material, indicating that the crystals likely resulted from a biologically-induced mineralization process. Although the hematite particles appear to consist of aggregations of many small (5 to 10 nm) grains, the grains have a consensus orientation and thus the whole particle diffracts as a single crystal. The aligned arrangement of nanoparticles within larger clusters may reflect either a templated nucleation of hematite crystallites in an extracellular organic matrix, or result from a self-assembling process during the crystallization of hematite from ferric gels or ferrihydrite

Crystal-size and shape distributions of magnetite from uncultured magnetotactic bacteria as a potential biomarker

We studied the sizes and shapes of magnetite nanocrystals produced by several types of uncultured magnetotactic bacteria to understand whether their size distributions can be used for identifying the biogenic origin of magnetite crystals in geological samples. The two-dimensional projections of the crystals were measured on transmission electron microscope (TEM) images, and features of crystal-size and shape-factor distributions (CSD and SFD, respectively) were analyzed. In agreement with previous results, most magnetite CSD curves are asymmetric and negatively skewed; however, one magnetotactic strain produced particles that have a normal size distribution. A statistical analysis of CSDs and SFDs (both from this and previous studies) reveals similarities among magnetite from magnetotactic strains from various locations. In particular, crystals in a cultured marine strain (MC-2) were indistinguishable from magnetite from a freshwater strain. We tested whether CSDs of distinct magnetosome types can be recovered from the shape and size data of all particles combined in samples that contain several types of magnetosomes; such samples can be used as models for rocks that contain magnetite nanocrystals of unknown and, presumably, various origins. If the SFDs of the distinct magnetosome types occurring in the same sample differ, the CSDs of individual magnetosome types can be retrieved from bulk data. In such cases the characteristic shape of the size distribution can be used for identifying magnetite as originating from magnetotactic bacteria