Validation of the Legionella pneumophila SG1 DETECT Kit for Quantification of Legionella pneumophila Serogroup 1 Bacteria in Potable Waters, Process Waters, and Surface Waters: AOAC Performance Tested MethodSM 052002.

The L.p.SG1 DETECT Kit is a rapid, quantitative method for the detection and enumeration of Legionella pneumophila serogroup 1 (L.p. SG1) bacteria from different water matrixes. The method is based on a combination of immunomagnetic separation (IMS) and flow cytometric (FCM) quantification. To this end, the method employs magnetic particles conjugated to anti-L.p. SG1 antibodies for the IMS of the target bacteria from environmental matrices and fluorescently labeled anti-L.p. SG1 antibodies for subsequent quantification by FCM. The IMS can be performed either manually with a magnetic rack (rqmicro.MIMS) or automated with the rqmicro.STREAM sample preparation instrument. Compared to the reference method ISO 11731:2017, which is based on culturing and enumeration of colony forming units (CFU) on agar plates, and can take up to 10 days until results are available, analysis with the L.p. SG1 DETECT Kit is culture-independent and delivers results within 2 h. This Performance Tested Method validation study demonstrates a robust method with recoveries exceeding 69%, inclusivity of 100%, exclusivity of 97.2%, and a shelf life of at least 6 months at 4°C or 40 days at 25°C. The Limit of Detection (LOD) was determined at 21 CFU/L and the Limit of Quantification (LOQ) at 80 CFU/L for potable water using the rqmicro.STREAM. The matrix study across three different types of water matrixes (potable, surface, and industrial process water), demonstrates superior repeatability and reproducibility, as well as equivalent or even superior detection of L.p. SG1 bacteria compared to the standard ISO 11731 method.

Engineered peptide barcodes for in-depth analyses of binding protein libraries.

Binding protein generation typically relies on laborious screening cascades that process candidate molecules individually. We have developed NestLink, a binder selection and identification technology able to biophysically characterize thousands of library members at once without the need to handle individual clones at any stage of the process. NestLink uses genetically encoded barcoding peptides termed flycodes, which were designed for maximal detectability by mass spectrometry and support accurate deep sequencing. We demonstrate NestLink's capacity to overcome the current limitations of binder-generation methods in three applications. First, we show that hundreds of binder candidates can be simultaneously ranked according to kinetic parameters. Next, we demonstrate deep mining of a nanobody immune repertoire for membrane protein binders, carried out entirely in solution without target immobilization. Finally, we identify rare binders against an integral membrane protein directly in the cellular environment of a human pathogen. NestLink opens avenues for the selection of tailored binder characteristics directly in tissues or in living organisms.

The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers.

Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers (e.g. viral capsid proteins). To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1ß in vitro. To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.

TRIM5 is an innate immune sensor for the retrovirus capsid lattice.

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Arginine-induced conformational change in the c-ring/a-subunit interface of ATP synthase.

The rotational mechanism of ATP synthases requires a unique interface between the stator a subunit and the rotating c-ring to accommodate stability and smooth rotation simultaneously. The recently published c-ring crystal structure of the ATP synthase of Ilyobacter tartaricus represents the conformation in the absence of subunit a. However, in order to understand the dynamic structural processes during ion translocation, studies in the presence of subunit a are required. Here, by intersubunit Cys-Cys cross-linking, the relative topography of the interacting helical faces of subunits a and c from the I. tartaricus ATP synthase has been mapped. According to these data, the essential stator arginine (aR226) is located between the c-ring binding pocket and the cytoplasm. Furthermore, the spatially vicinal residues cT67C and cG68C in the isolated c-ring structure yielded largely asymmetric cross-linking products with aN230C of subunit a, suggesting a small, but significant conformational change of binding-site residues upon contact with subunit a. The conformational change was dependent on the positive charge of the stator arginine or the aR226H substitution. Energy-minimization calculations revealed possible modes for the interaction between the stator arginine and the c-ring. These biochemical results and structural restraints support a model in which the stator arginine operates as a pendulum, moving in and out of the binding pocket as the c-ring rotates along the interface with subunit a. This mechanism allows efficient interaction between subunit a and the c-ring and simultaneously allows almost frictionless movement against each other.