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

Abstract

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.

Unbiased Identification of Angiogenin as an Endogenous Antimicrobial Protein With Activity Against Virulent Mycobacterium tuberculosis

“…Only few AMPs with activity against virulent Mtb have been described, which may be related to the complexity of the lipid-rich mycobacterial cell wall, the slow metabolism/generation time of Mtb or simply due to the requirements for a biological safety level 3 laboratory. Granulysin ( Stenger et al, 1998 ), human neutrophil peptides, such as human ß-defensins ( Linde et al, 2001 ), Protegrin 1 ( Fattorini et al, 2004 ), Lipocalin 1 ( Martineau et al, 2007 ), LL-37 ( Deshpande et al, 2020 ), Lassomycin ( Gavrish et al, 2014 ), Teixobactin ( Ling et al, 2015 ), or Hepcidin ( Sow et al, 2007 ) act at fairly high concentrations ranging between 10 and 50 μM, which is within the same range as we observed for Angiogenin and Angie1 ( Figures 2 , 5 ). Generally, cationic AMPs interact with the negatively charged mycomembrane and plasma membrane of Mtb ( Gutsmann, 2016 ).…”

“…Studies on the mechanisms of action of these AMPs are limited since Mtb has a notoriously low metabolism, long generation time and is a biosafety level 3 pathogen. Imaging of AMP-treated Mtb indicates that Granulysin and LL-37 disrupt the mycobacterial cell wall ( Stenger et al, 1998 ; Deshpande et al, 2020 ). It is unclear whether this is the lethal hit or only the initial step to allow the entry into the bacterial cytosol to reach its final intracellular target.…”

Delivery by Dendritic Mesoporous Silica Nanoparticles Enhances the Antimicrobial Activity of a Napsin‐Derived Peptide Against Intracellular Mycobacterium tuberculosis

“…[13] This amphiphilic structural feature seems to be relevant for the postulated mode of action of the peptides, which is the penetration of the bacterial cell membrane by several proposed mechanisms. [14][15][16][17] As Mtb is an obligate intracellular bacterium and by the chemical nature of AMPs some of the most important barriers in the therapeutic application are the limited cellular uptake of the peptide, the short circulation time and their low stability in body fluids and the intracellular environment due to excretion and enzymatic degradation. [18,19] In order to overcome these limitations, one major opportunity is the encapsulation of the therapeutic peptide within a suitable (nano-) carrier system, like vesicular structures (liposomes) and polymeric or inorganic nanoparticles.…”

Cyanobacteria and Eukaryotic Microalgae as Emerging Sources of Antibacterial Peptides

“…In addition to the development in synthesis, there are also advances in peptide purification and analysis methodologies that include techniques, such as HPLC, MS, circular dichroism (CD), and nuclear magnetic resonance (NMR), which can be helpful in studies of structure-activity relationship or in the determination of mechanisms of action [ 184 , 199 , 200 , 201 ].…”