The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria

Abstract

Cell-autonomous innate immune responses against bacteria attempting to colonize the cytosol of mammalian cells are incompletely understood. Polyubiquitylated proteins can accumulate on the surface of such bacteria, and bacterial growth is restricted by Tank-binding kinase (TBK1). Here we show that NDP52, not previously known to contribute to innate immunity, recognizes ubiquitin-coated Salmonella enterica in human cells and, by binding the adaptor proteins Nap1 and Sintbad, recruits TBK1. Knockdown of NDP52 and TBK1 facilitated bacterial proliferation and increased the number of cells containing ubiquitin-coated salmonella. NDP52 also recruited LC3, an autophagosomal marker, and knockdown of NDP52 impaired autophagy of salmonella. We conclude that human cells utilize the ubiquitin system and NDP52 to activate autophagy against bacteria attempting to colonize their cytosol.

Recruitment of TBK 1 to cytosol‐invading Salmonella induces WIPI 2‐dependent antibacterial autophagy

“…To substantiate this finding, we next investigated where in the anti‐bacterial autophagy pathway TBK1 acts. By complementing Tbk1 −/− MEFs, we confirmed that lack of TBK1 increased the percentage of ubiquitin‐coated cytosolic S.  Typhimurium at 4 h post‐infection (Fig 1C) (Radtke et al , 2007; Thurston et al , 2009). TBK1 K38M and TBK1 ΔC , which are catalytically inactive and deficient in binding adaptor proteins, respectively, did not complement the ubiquitin phenotype, in line with the lack of these alleles to control proliferation of S.  Typhimurium in the cytosol of host cells (Fig 1A and C).…”

“…Typhimurium in Tbk1 −/− MEFs, a phenotype complemented with wild‐type but not catalytically inactive TBK1 K38M (Figs 1A and EV1A) (Pomerantz & Baltimore, 1999; Radtke et al , 2007). We have previously shown that TBK1 physically associates with those intracellular Salmonella that are positive for the TBK1 adaptor proteins Nap1 and Sintbad and the autophagy cargo receptor NDP52 (Thurston et al , 2009). To test whether the function of TBK1 in anti‐bacterial autophagy requires interactions with its adaptor proteins we truncated TBK1 at its C‐terminus (TBK1 N685 hereafter referred to as TBK1 ΔC ), thereby generating a molecule deficient in binding to all its known adaptors, that is Nap1, Sintbad, Tank, and optineurin (Fig EV1B) (Goncalves et al , 2011), while maintaining kinase activity as indicated by the activation of an ISRE reporter (Fig EV1C).…”

“…VPS34 produces membrane patches rich in phosphatidylinositol 3‐phosphate (PI(3)P) that recruit PI(3)P‐binding proteins such as WIPI and DFCP1 to the site of phagophore formation (Axe et al , 2008). In contrast to the non‐selective engulfment of cytosol into starvation‐induced autophagosomes, anti‐bacterial autophagy is mediated by cargo receptors including NDP52, optineurin, and p62 (Thurston et al , 2009; Zheng et al , 2009; Wild et al , 2011). Cargo receptors bind members of the LC3/GABARAP family of ubiquitin‐like proteins on the autophagosomal membrane and specific “eat‐me” signals associated with cytosol‐invading bacteria, thereby selectively tethering bacteria to phagophore membranes (Weidberg et al , 2011; Rogov et al , 2014).…”

“…Typhimurium also requires the kinase TBK1, a member of the IKK (inhibitor of nuclear factor κB kinase) family (Radtke et al , 2007; Thurston et al , 2009). The anti‐bacterial function of TBK1 is distinct from its well‐characterized role of inducing type I interferons by phosphorylating IRF3 in virally infected cells (Randow et al , 2013; Wu & Chen, 2014).…”

“…The anti‐bacterial function of TBK1 is distinct from its well‐characterized role of inducing type I interferons by phosphorylating IRF3 in virally infected cells (Randow et al , 2013; Wu & Chen, 2014). TBK1 accumulates in the vicinity of cytosol‐exposed bacteria together with its adaptor proteins Nap1, Sintbad, and their binding partner NDP52 (Fujita et al , 2003; Ryzhakov & Randow, 2007; Thurston et al , 2009; Verlhac et al , 2015). TBK1 also associates with optineurin and it has been reported to phosphorylate both optineurin and p62, thereby enhancing their affinity for LC3B and ubiquitin, respectively (Morton et al , 2008; Wild et al , 2011; Pilli et al , 2012; Heo et al , 2015; Richter et al , 2016).…”