The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3.

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at serine 10 and serine 28. We show that CbFic2 acts as a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via a C-terminal helix-turn-helix domain. We propose that CbFic2 performs AMPylation in its monomeric state, switching to a deAMPylating dimer upon DNA binding. This study unveils reversible histone modification by a specific enzyme of a pathogenic bacterium.

Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.

The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions.

Legionella spp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different "effector" proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays in Legionella longbeachae lysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)P binding of SidC] fragment. Isothermal titration calorimetry revealed that SidC of L. longbeachae (SidC(Llo)) binds PtdIns(4)P with a K(d) (dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue of Legionella pneumophila (SidC(Lpn)). Upon infection of RAW 264.7 macrophages with L. longbeachae, endogenous SidC(Llo) or ectopically produced SidC(Lpn) localized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. An L. longbeachae ΔsidC deletion mutant was impaired for calnexin recruitment to LCVs in Dictyostelium discoideum amoebae and outcompeted by wild-type bacteria in Acanthamoeba castellanii. Calnexin recruitment was restored by SidC(Llo) or its orthologues SidC(Lpn) and SdcA(Lpn). Conversely, calnexin recruitment was restored by SidC(Llo) in L. pneumophila lacking sidC and sdcA. Together, biochemical, genetic, and cell biological data indicate that SidC(Llo) is an L. longbeachae effector that binds through a P4C domain with high affinity to PtdIns(4)P on LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.

Protein LidA from Legionella is a Rab GTPase supereffector.

The causative agent of Legionnaires disease, Legionella pneumophila, injects several hundred proteins into the cell it infects, many of which interfere with or exploit vesicular transport processes. One of these proteins, LidA, has been described as a Rab effector (i.e., a molecule that interacts preferentially with the GTP-bound form of Rab). We describe here the structure and biochemistry of a complex between the Rab-binding domain of LidA and active Rab8a. LidA displays structural peculiarities in binding to Rab8a, forming a considerably extended interface in comparison to known mammalian Rab effectors, and involving regions of the GTPase that are not seen in other Rab:effector complexes. In keeping with this extended binding interface, which involves four α-helices and two pillar-like structures of LidA, the stability of LidA-Rab interactions is dramatically greater than for other such complexes. For Rab1b and Rab8a, these affinities are extraordinarily high, but for the more weakly bound Rab6a, K(d) values of 4 nM for the inactive and 30 pM for the active form were found. Rab1b and Rab8a appear to bind LidA with K(d) values in the low picomolar range, making LidA a Rab supereffector.