Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

No abstract

Urinary tract infections (UTIs) cause a substantial health care burden. UTIs (i) are most often caused by uropathogenic Escherichia coli (UPEC), (ii) primarily affect otherwise healthy females (50% of women will have a UTI), (iii) are associated with significant morbidity and economic impact, (iv) can become chronic, and (v) are highly recurrent. A history of UTI is a significant risk factor for a recurrent UTI (rUTI). In otherwise healthy women, an acute UTI leads to a 25 to 50% chance of rUTI within months of the initial infection. Interestingly, rUTIs are commonly caused by the same strain of E. coli that led to the initial infection, arguing that there exist host-associated reservoirs, like the gastrointestinal tract and underlying bladder tissue, that can seed rUTIs. Additionally, catheter-associated UTIs (CAUTI), caused by Enterococcus and Staphylococcus as well as UPEC, represent a major health care concern. The host’s response of depositing fibrinogen at the site of infection has been found to be critical to establishing CAUTI. The Drug Resistance Index, an evaluation of antibiotic resistance, indicates that UTIs have become increasingly difficult to treat since the mid-2000s. Thus, UTIs are a “canary in the coal mine,” warning of the possibility of a return to the preantibiotic era, where some common infections are untreatable with available antibiotics. Numerous alternative strategies for both the prevention and treatment of UTIs are being pursued, with a focus on the development of vaccines and small-molecule inhibitors targeting virulence factors, in the hopes of reducing the burden of urogenital tract infections in an antibiotic-sparing manner.

Chaperone-usher pathway (CUP) pili are extracellular proteinaceous fibers ubiquitously found on Gram-negative bacteria, and mediate host-pathogen interactions and biofilm formation critical in pathogenesis in numerous human diseases 1 . During pilus assembly an outer membrane (OM) macromolecular machine called the usher catalyzes pilus biogenesis from the individual subunits that are delivered as chaperone-subunit complexes in the periplasm. The usher orchestrates pilus assembly using all five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PD), an amino-terminal periplasmic domain (NTD) and two carboxy-terminal periplasmic domains (CTD1 and CTD2) 2 – 6 . Despite extensive structural and functional characterization, the mechanism by which the usher is activated to initiate pilus biogenesis is unknown. Here we present the crystal structure of the full-length PapC usher from Escherichia coli in complex with its cognate PapDG chaperone-subunit complex in a pre-activation state, elucidating molecular details of how the usher is specifically engaged by allosteric interactions with its substrate preceding activation and how the usher facilitates the transfer of subunits from the NTD to the CTDs during pilus assembly. This work elucidates the intricate workings of a molecular machine that catalyzes CUP pilus assembly and opens the door for the development of potent inhibitors to block pilus biogenesis.