Since GAS bacteria secrete SLO to disrupt the endosomal membrane (Nakagawa et al., 2004) and ubiquitinated cytoplasmic GAS bacteria are targeted by autophagy (von Muhlinen et al., 2010), we used Galectin-3, a marker of damaged endomembranes (Paz et al., 2010), and a FK2 polyubiquitin antibody. The position of the expected PCR amplicon is usually indicated by an arrowhead. Image_1.JPEG (587K) GUID:?2D20CDFA-4369-4E8D-A1E8-78533AF018D7 Supplementary Figure S2: Generation of Beclin 1, UVRAG, and Rubicon KO cells using the CRISPR/Cas9 system and knockdown (KD) of Vps34 and Atg14. (ACC) HeLa wild-type and either Beclin 1 (A), UVRAG (B), or Rubicon (C) KO cells were analyzed by immunoblotting using corresponding antibodies. Sequences of the wild-type Beclin 1, UVRAG, or Rubicon locus and mutated allele of each KO cell line around the target locus. The targeted locus of gRNA and the protospacer-adjacent motif (PAM) sequences are indicated by underline and red letters, respectively. Deleted nucleotides are indicated by hyphens. (D,E) Immunoblotting analysis of Vps34 (D) and Atg14 (E) knockdown HeLa cells. HeLa cells were transfected with either control siRNA, or siRNA targeting Vps34 or Atg14. Expression of Vps34 and Atg14 was analyzed by western blotting using corresponding antibodies. Image_2.JPEG (423K) GUID:?90D65365-5260-4562-B49D-8BAABBD7E175 Supplementary Figure S3: Construction of NLRX1 deletion mutants. (A) Schematic representation of NLRX1 deletion mutants. (B) The expression profile of deletion mutants was determined by western blotting using an anti-FLAG antibody. (C) Confocal micrographs of HeLa cells transfected with EmGFP-tagged NLRX1 deletion mutants. Mitochondria and nuclei were stained with MitoTacker dye and DAPI, respectively. Scale bars, 10 m. Image_3.JPEG (610K) GUID:?48918237-3641-49D4-8E04-E2B28AA1A8B2 Abstract Group A (GAS) can invade epithelial cells; however, these bacteria are targeted and eventually destroyed by autophagy. Members of the Nod-like receptor (NLR) family are thought to be critical for the autophagic LOR-253 response to invasive bacteria. However, the intracellular sensors within host cells that are responsible for bacterial invasion and the induction of autophagy are largely unknown. Thus, our aim was to examine the role of one such NLR, namely NLRX1, in invasion and autophagy during GAS contamination. We found that GAS invasion was markedly increased in NLRX1 knockout cells. This led to the potentiation of autophagic processes such as autophagosome and autolysosome formation. NLRX1 was found to interact with Beclin 1 and UVRAG, members of Beclin1 complex, and knockout of these proteins LOR-253 inhibited invasion and autophagy upon GAS contamination. Especially, NLRX1 interacted with Beclin 1 via its NACHT domain name and this conversation was responsible for the NLRX1-mediated inhibition of invasion and autophagic processes including autophagosome and autolysosome formation during GAS contamination. These findings demonstrate that NLRX1 functions as a negative regulator to inactivate the Beclin 1CUVRAG complex, which regulates invasion and autophagy during GAS contamination. Thus, our study expands our knowledge of the role of NLRX1 during bacterial invasion and autophagy and could lead to further investigations to understand pathogenChost cell interactions, facilitating novel targeted therapeutics. (GAS; and into autophagosomes (Travassos et al., 2010). In addition, some NLRs such as NLRC4 and NLRP4 were shown to associate with Beclin 1, which in turn negatively regulates autophagy during bacterial infection (Jounai et al., 2011). However, the involvement of the NLRX1CBeclin 1 complex in autophagy in response to bacterial infection remains unknown. In this study, we examined the role of NLRX1 in invasion and autophagy during GAS contamination, and showed that NLRX1 inhibits endocytosis-mediated invasion of GAS bacteria into host epithelial cells, which consequently results in the suppression of autophagy to clear cytoplasmic GAS. Notably, these inhibitory effects on Icam4 invasion and autophagy were attributed to the conversation between NLRX1 and the Beclin 1CUVRAG complex. Materials and methods Cell culture and transfection HeLa cells were purchased from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (Gibco) and 50 g/mL gentamicin (Nacalai Tesque) in a 5% CO2 incubator at 37C. Plasmid transfections were performed using polyethylenimine (Polysciences, Inc.), Lipofectamine 3000 (Invitrogen), or Lipofectamine RNAiMAX (Invitrogen), according to the manufacturers’ protocols. Group A strain Group A (GAS) LOR-253 strain JRS4 (M6+ F1+) was grown in ToddCHewitt broth (BD Diagnostic Systems, Sparks, MD) supplemented with 0.2% yeast extract (THY), as described previously (Nakagawa et al., 2004). Plasmid construction Gateway cloning technology (Invitrogen) was used to create the vectors indicated as follows. Human (GenBank Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_024618.3″,”term_id”:”531034768″,”term_text”:”NM_024618.3″NM_024618.3), (GenBank Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002647.3″,”term_id”:”808688272″,”term_text”:”NM_002647.3″NM_002647.3), (GenBank Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003766.4″,”term_id”:”929524265″,”term_text”:”NM_003766.4″NM_003766.4), ATG14 (GenBank Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_014924.4″,”term_id”:”335057541″,”term_text”:”NM_014924.4″NM_014924.4), and (GenBank Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003369.3″,”term_id”:”111160877″,”term_text”:”NM_003369.3″NM_003369.3) were PCR-amplified from human cDNA libraries using the.