Type IV pili play important roles in a wide array of processes, including surface adhesion and twitching motility. Although archaeal genomes encode a diverse set of type IV pilus subunits, the functions for most remain unknown. We have now characterized six Haloferax volcanii pilins, PilA[1-6], each containing an identical 30-amino-acid N-terminal hydrophobic motif that is part of a larger highly conserved domain of unknown function (Duf1628). Deletion mutants lacking up to five of the six pilin genes display no significant adhesion defects; however, H. volcanii lacking all six pilins (⌬pilA[1-6]) does not adhere to glass or plastic. Consistent with these results, the expression of any one of these pilins in trans is sufficient to produce functional pili in the ⌬pilA[1-6] strain. PilA1His and PilA2His only partially rescue this phenotype, whereas ⌬pilA[1-6] strains expressing PilA3His or PilA4His adhere even more strongly than the parental strain. Most surprisingly, expressing either PilA5His or PilA6His in the ⌬pilA[1-6] strain results in microcolony formation. A hybrid protein in which the conserved N terminus of the mature PilA1His is replaced with the corresponding N domain of FlgA1 is processed by the prepilin peptidase, but it does not assemble functional pili, leading us to conclude that Duf1628 can be annotated as the N terminus of archaeal PilA adhesion pilins. Finally, the pilin prediction program, FlaFind, which was trained primarily on archaeal flagellin sequences, was successfully refined to more accurately predict pilins based on the in vivo verification of PilA[1-6].

INTRODUCTION: Cerebrospinal fluid (CSF) biomarkers can identify individuals withAlzheimer disease (AD) pathology (e.g., amyloid plaques, neurofibrillary tangles), but defined analyte cut-points using high-throughput automated assays are necessary for general clinical use. METHODS: CSF Aβ42, t-tau, and t-tau/Aβ42 were quantified by the LUMIPULSE platform in two test cohorts (A/B: Eisai BAN2401-201/MISSION AD E2609-301/302, n=138; C: Knight AD Research Center, n=198), and receiver operating characteristic (ROC) curve analyses defined cutpoints corresponding best to amyloid determinations using PET imaging. The best-performing cutpoint was then validated as a predictor of amyloid status in an independent cohort (D: MISSION AD E2609-301/302; n=240). RESULTS:Virtually identical t-tau/Aβ42 cut-points (~0.54) performed best in both test cohorts and with similar accuracy (areas under ROC curve [AUC] (A/B: 0.95; C: 0.94). The cut-point yielded an overall percent agreement with amyloid PET of 85.0% in validation cohort D. DISCUSSION: LUMIPULSE CSF biomarker measures with validated cut-points have clinical utility in identifying AD pathology.

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SUMMARY Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings.

Type IV pili are ancient proteinaceous structures present on the cell surface of species in nearly all bacterial and archaeal phyla. These filaments, which are required for a diverse array of important cellular processes, are assembled employing a conserved set of core components. While type IV pilins, the structural subunits of pili, share little sequence homology, their signal peptides are structurally conserved allowing for in silico prediction. Recently, in vivo studies in model archaea representing the euryarchaeal and crenarchaeal kingdoms confirmed that several of these pilins are incorporated into type IV adhesion pili. In addition to facilitating surface adhesion, these in vivo studies also showed that several predicted pilins are required for additional functions that are critical to biofilm formation. Examples include the subunits of Sulfolobus acidocaldarius Ups pili, which are induced by exposure to UV light and promote cell aggregation and conjugation, and a subset of the Haloferax volcanii adhesion pilins, which play a critical role in microcolony formation while other pilins inhibit this process. The recent discovery of novel pilin functions such as the ability of haloarchaeal adhesion pilins to regulate swimming motility may point to novel regulatory pathways conserved across prokaryotic domains. In this review, we will discuss recent advances in our understanding of the functional roles played by archaeal type IV adhesion pili and their subunits, with particular emphasis on their involvement in biofilm formation.