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.

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.

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

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 C3bot_E174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3bot_E174Q 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 C3bot_E174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3bot_E174Q 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 C3bot_E174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.