Stanimir Ivanov

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA.

CpG‐DNA and its related synthetic CpG oligodeoxynucleotides (CpG‐ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG‐DNA‐responsive serine/threonine kinases; however, the target protein of CpG‐DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow‐derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) results in defective phosphorylation and activation of Akt by CpG‐DNA. Unexpectedly, loss of the Toll‐like receptor 9 has a minimal effect on Akt activation in response to CpG‐DNA. Further in vitro analysis using purified DNA‐PK and recombinant Akt proteins reveals that DNA‐PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA‐PKcs associates with Akt upon CpG‐DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA‐PKcs in CpG‐DNA signaling and define a CpG‐DNA/DNA‐PKcs/Akt pathway.

Lipidation by the host prenyltransferase machinery facilitates membrane localization of Legionella pneumophila effector proteins.

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.

Activation of IKK by thymosin α1 requires the TRAF6 signalling pathway

Thymosin α1 (Tα1) is noted for its immunomodulatory activities and therapeutic potential in treatment of infectious diseases and cancer. However, the molecular mechanism of its effectiveness is not completely understood. Here, we report that Tα1 induces interleukin (IL)‐6 expression through the IκB kinase (IKK) and nuclear factor‐κB (NF‐κB) pathway. Using IKKβ‐deficient bone‐marrow‐derived macrophages and mouse embryo fibroblasts (MEFs), we show that IKKβ is essential for IKK and NF‐κB activation as well as efficient IL‐6 induction. Further analysis using tumour necrosis factor receptor‐associated factor 6 (TRAF6)‐deficient MEFs shows that TRAF6 is crucial for activation of IKK and induction of IL‐6 by Tα1. Intriguingly, Tα1 triggers protein kinase C (PKC)ι/ζ activation, which is TRAF6 dependent and involves IKK. In addition, Tα1 induces the formation of a signalsome composed of TRAF6, p62 and PKCι/ζ as well as IKK. Thus, our study identifies Tα1 as a unique activator of the TRAF6 signal pathway and provides a cohesive interpretation of the molecular basis of the therapeutic utility of Tα1.

Pathogen signatures activate a ubiquitination pathway that modulates the function of the metabolic checkpoint kinase mTOR

The mammalian immune system has the ability to discriminate between pathogenic microbes and nonpathogenic microbes to control inflammation. Here we investigated the ubiquitination profiles of host proteins after infection of macrophages with a virulent strain of the intracellular bacterium Legionella pneumophila or a nonpathogenic mutant of L. pneumophila. Only infection with pathogenic L. pneumophila resulted in ubiquitination of positive regulators of the metabolic checkpoint kinase mTOR and led to diminished mTOR activity. Detection of pathogen signatures resulted in translational biasing toward proinflammatory cytokines through mTOR-mediated regulation of cap-dependent translation. Thus, there is a pathogen-detection program in macrophages that stimulates protein ubiquitination and the degradation of regulators of mTOR, which suppresses mTOR function and directs a proinflammatory cytokine program.

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly‐ubiquitin conjugates to proteins associated with L. pneumophila‐containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila‐derived molecules resulted in the production of ubiquitin‐rich dendritic cell aggresome‐like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin‐decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.

NDP52: the missing link between ubiquitinated bacteria and autophagy

Mammalian cells ubiquitinate bacteria that erroneously enter the cytosol and target these intruding microbes for destruction by autophagy. New work shows that the protein NDP52 directly binds to ubiquitinated bacteria and facilitates the assembly of an autophagic membrane that surrounds these invaders.

Host lipidation: a mechanism for spatial regulation of Legionella effectors.

Bacterial pathogens have evolved the capacity to translocate proteins into the cytosol of infected cells to manipulate host processes. How do pathogens regulate spatially these bacterial effector proteins once they are released into the host cell? One mechanism, which is used by Legionella and other bacterial pathogens, is to encode effectors that mimic the substrates of eukaryotic lipid transferases. In this review we discuss three membrane-targeting pathways in eukaryotes that are exploited by Legionella and other pathogens-prenylation, palmitoylation, and myristoylation. Lipidation of bacterial substrates primes the effectors for coincidence detection-mediated targeting onto membrane-bound organelles by increasing membrane affinity. Intracellular membrane-targeting strategies that exploit protein fatty acylation and prenylation direct bacterial effectors to compartments where their target substrates reside and thus are critical for effector function.

MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis

Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.