TLR-mediated aggresome-like induced structures comprise antimicrobial peptides and attenuate intracellular bacterial survival

Abstract

Abstract Immune cells employ diverse mechanisms for host defense against pathogens. Macrophages and dendritic cells, in response to toll-like receptor (TLR) activation, assemble aggresome-like induced structures (ALIS). Our group has previously shown that engagement of TLR4 transcriptionally upregulates p62/sequestome1, which in turn assembles ALIS along with LC3 and ubiquitin. We have demonstrated that TLR4 mediated autophagy is, in fact, selective autophagy of ALIS. We hypothesize that TLR-mediated autophagy and ALIS contribute to host-defense. Here we show that ALIS are not only assembled in macrophages upon exposure to several types of bacteria, but these structures are associated with pathogen-containing phagosomes. Importantly, we also present evidence of increased bacterial burden in cells, in which formation of ALIS is prevented with p62 specific siRNA. To gain more insight into the assembly of the constituents of ALIS, we have employed 3D super-resolution structured illumination microscopy (3D SR-SIM) and mass-spectrometric analyses. Ultra-structural analyses by 3D SR-SIM of known constituents of ALIS (namely p62, ubiquitin and LC3) reveals that ALIS are organized structures with distinct patterns of alignment. Furthermore, mass spectrometric analyses of ALIS identified, among others, several proteins of known antimicrobial properties. We have validated mass spectrometry analysis data by testing the association of some of these molecules (Bst2, IFITM2 and IFITM3) with ALIS and the phagocytosed bacteria. Thus, we surmise the enrichment of AMPs in ALIS leads to their delivery to bacteria-containing phagosomes and to restrict the bacteria in host cells. Our findings in this paper support hitherto unknown functions of ALIS in host-defense.