Orientia tsutsugamushi Ank9 is a multifunctional effector that utilizes a novel GRIP-like Golgi localization domain for Golgi-to-endoplasmic reticulum trafficking and interacts with host COPB2.

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that afflicts 1 million people annually. This obligate intracellular bacterium boasts one of the largest microbial arsenals of ankyrin repeat-containing protein (Ank) effectors, most of which target the endoplasmic reticulum (ER) by undefined mechanisms. Ank9 is the only one proven to function during infection. Here, we demonstrate that Ank9 bears a motif that mimics the GRIP domain of eukaryotic golgins and is necessary and sufficient for its Golgi localization. Ank9 reaches the ER exclusively by retrograde trafficking from the Golgi. Consistent with this observation, it binds COPB2, a host protein that mediates Golgi-to-ER transport. Ank9 destabilizes the Golgi and ER in a Golgi localization domain-dependent manner and induces the activating transcription factor 4-dependent unfolded protein response. The Golgi is also destabilized in cells infected with O. tsutsugamushi or treated with COPB2 small interfering RNA. COPB2 reduction and/or the cellular events that it invokes, such as Golgi destabilization, benefit Orientia replication. Thus, Ank9 or bacterial negative modulation of COPB2 might contribute to the bacterium's intracellular replication. This report identifies a novel microbial Golgi localization domain, links Ank9 to the ability of O. tsutsugamushi to perturb Golgi structure, and describes the first mechanism by which any Orientia effector targets the secretory pathway.

Structural and mechanistic features of protein O glycosylation linked to CD8+ T-cell apoptosis.

CD8+ T-cell apoptosis is essential for the contraction phase of the immune response, yet the initiating signals and precise pathways involved are unresolved. The ST3Gal-I sialyltransferase is a candidate mechanistic component and catalyzes sialic acid addition to core 1 O-glycans during protein O glycosylation. ST3Gal-I inactivation or enzymatic removal of its product renders CD8+ T cells, but not CD4+ T cells, susceptible to apoptosis by differential cross-linking of O-glycoproteins in the absence of interleukin-2 and T-cell receptor (TCR) signaling. This results in caspase activation, DNA fragmentation, and phosphatidylserine externalization prior to cell death. We further show that ST3Gal-I function is regulated by a posttranscriptional mechanism operating distal to Golgi core 2 O glycosylation and is invariably linked to CD8+ T-cell contraction following viral (lymphocytic choriomeningitis virus) infection and bacterial (staphylococcal enterotoxin B) antigen immunization. The mechanism does not involve the ST3Gal-I substrate CD43 or core 2 O-glycan induction and overcomes the ability of Bcl-2 to inhibit the contraction phase in vivo. Loss of ST3Gal-I function further reduces Bim-deficient CD8+ T-cell accumulation without diminishing apoptotic sensitivity. We propose that an endogenous lectin activates an apoptotic pathway constructed in CD8+ T cells following TCR stimulation and enables contraction upon attenuation of immune signaling.

ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling.

The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation.