Helmholtz Zentrum für Infektionsforschung Repository
Not a member yet
4806 research outputs found
Sort by
Ly6G deficiency alters the dynamics of neutrophil recruitment and pathogen capture during Leishmania major skin infection.
No full textNeutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils' ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system
Within-Host Adaptation of in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity.
No full textWithin-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen's strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype-phenotype associations under different infection-relevant growth conditions
Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1.
No full textThe application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing
BRD9 is a druggable component of interferon-stimulated gene expression and antiviral activity.
No full textInterferon (IFN) induction of IFN-stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG upregulation. Here, we used genome-scale loss-of-function screening to establish genes critical for IFN-induced transcription, identifying all expected members of the JAK-STAT signaling pathway and a previously unappreciated epigenetic reader, bromodomain-containing protein 9 (BRD9), the defining subunit of non-canonical BAF (ncBAF) chromatin-remodeling complexes. Genetic knockout or small-molecule-mediated degradation of BRD9 limits IFN-induced expression of a subset of ISGs in multiple cell types and prevents IFN from exerting full antiviral activity against several RNA and DNA viruses, including influenza virus, human immunodeficiency virus (HIV1), and herpes simplex virus (HSV1). Mechanistically, BRD9 acts at the level of transcription, and its IFN-triggered proximal association with the ISG transcriptional activator, STAT2, suggests a functional localization at selected ISG promoters. Furthermore, BRD9 relies on its intact acetyl-binding bromodomain and unique ncBAF scaffolding interaction with GLTSCR1/1L to promote IFN action. Given its druggability, BRD9 is an attractive target for dampening ISG expression under certain autoinflammatory conditions
Helical reconstruction of and needle filaments attached to type 3 basal bodies.
No full textGram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development
Total Synthesis of Thuggacin cmc-A and Its Structure Determination.
No full textThe first total synthesis of thuggacin cmc-A and the determination of the absolute structure are described. The thuggacin family of antibiotics is of great interest due to the antibiotic activity against Mycobacterium tuberculosis. Based on the assumption that seven stereogenic centers in thuggacin cmc-A would share the same stereochemistry as thuggacin-A, all stereogenic centers of thuggacin cmc-A were strictly constructed in a stereocontrolled manner. The total synthesis allowed its stereostructure to be fully confirmed
Complete Genome Sequencing Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes
No full textThe ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence
Structural basis of the complete poxvirus transcription initiation process.
No full textPoxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription
Retiboletus (Boletaceae) in northern Thailand: one novel species and two first records
No full textMorphological characters and multi-gene phylogenetic analyses were used to identify Retiboletus specimens collected in northern Thailand. Retiboletus brevibasidiatus is described as new to science, whereas R. fuscus and R. nigrogriseus are reported for the first time from Thailand. Retiboletus brevibasidiatus produces medium-sized basidiomes, with a dark blonde to clay pileus and densely reticulate stipe mostly on the upper part with pale yellow to chrome yellow basal mycelium. It is difficult to separate R. brevibasidiatus from other closely related species on the basis of macroscopic characters. However, the new species can be distinguished by microscopic characters, mostly the shorter basidia. The macro- and micro-morphology of the R. fuscus and R. nigrogriseus collections from Thailand fit well with the previous descriptions of materials from China and Japan. Detailed descriptions, molecular phylogeny, and illustrations of the three species are provided
A Promiscuous Halogenase for the Derivatization of Flavonoids.
No full textHalogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper