ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

The Clostridium botulinum C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells.

The binary Clostridium (C.) botulinum C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA63. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins' catalytic A subunits. C2IIa as well as PA63 deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA63, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA63. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency.

Clostridium botulinum C3 Toxin for Selective Delivery of Cargo into Dendritic Cells and Macrophages.

The protein toxin C3bot from Clostridium botulinum is a mono-ADP-ribosyltransferase that selectively intoxicates monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (DCs) by cytosolic modification of Rho-A, -B, and -C. Here, we investigated the application of C3bot as well as its non-toxic variant C3botE174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3botE174Q facilitated the uptake of eGFP into early endosomes of human-monocyte-derived macrophages, as revealed by stimulated emission depletion (STED) super-resolution microscopy. The fusion of the cargo model peptide eGFP neither affected the cell-type selectivity (enhanced uptake into human macrophages ex vivo compared to lymphocytes) nor the cytosolic release of C3bot. Moreover, by cell fractionation, we demonstrated that C3bot and C3botE174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3botE174Q for covalent linkage of cargos via thiol-maleimide click chemistry. The functionality of this system was proven by loading small molecule fluorophores or an established reporter enzyme and investigating the cellular uptake and cytosolic release of cargo. Taken together, non-toxic C3botE174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.

Gran1: A Granulysin-Derived Peptide with Potent Activity against Intracellular Mycobacterium tuberculosis.

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed "Gran1"). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.

The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro.

The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.

Super-Resolution Microscopy Reveals a Direct Interaction of Intracellular Mycobacterium tuberculosis with the Antimicrobial Peptide LL-37.

The antimicrobial peptide LL-37 inhibits the growth of the major human pathogen Mycobacterium tuberculosis (Mtb), but the mechanism of the peptide-pathogen interaction inside human macrophages remains unclear. Super-resolution imaging techniques provide a novel opportunity to visualize these interactions on a molecular level. Here, we adapt the super-resolution technique of stimulated emission depletion (STED) microscopy to study the uptake, intracellular localization and interaction of LL-37 with macrophages and virulent Mtb. We demonstrate that LL-37 is internalized by both uninfected and Mtb infected primary human macrophages. The peptide localizes in the membrane of early endosomes and lysosomes, the compartment in which mycobacteria reside. Functionally, LL-37 disrupts the cell wall of intra- and extracellular Mtb, resulting in the killing of the pathogen. In conclusion, we introduce STED microscopy as an innovative and informative tool for studying host-pathogen-peptide interactions, clearly extending the possibilities of conventional confocal microscopy.

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells.

C3 protein toxins produced by Clostridium (C.) botulinum and C. limosum are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C. botulinum C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.

Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro.

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197's transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197.

Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core-Shell Matrixes.

Polymer particles with antibody-like affinity, i.e., molecularly imprinted polymers, offer an ideal platform for biopharmaceutical virus purification. In recent years, attempts combining molecular imprinting technology with a variety of visualization and detection techniques have been reported for directly confirming the localized presence of the template. Direct target visualization is crucial for the characterization of molecularly imprinted polymers, especially if biological templates such as viruses are used. In the present study, for the first time the viral binding behavior at virus-imprinted polymers (VIPs) via stimulated emission depletion (STED) microscopy is shown by imaging individual, fluorescently labeled virus particles. STED microscopy achieves among various other super-resolution techniques the best temporal resolution at high spatial resolution. An innovative virus purification material selective for human adenovirus type 5 (AdV5) offered highly purified virus for the subsequent fluorescent labeling procedure, thus enabling STED imaging. Excellent binding affinities (150-fold higher versus control particles) and high selectivity toward the target virus (AdV5) were observed at those VIPs, even in competitive binding experiments with minute virus of mice using dual-label STED microscopy.