Novel electronic biosensor for automated inoculum preparation to accelerate antimicrobial susceptibility testing.

A key predictor of morbidity and mortality for patients with a bloodstream infection is time to appropriate antimicrobial therapy. Accelerating antimicrobial susceptibility testing from positive blood cultures is therefore key to improving patient outcomes, yet traditional laboratory approaches can require 2-4 days for actionable results. The eQUANT-a novel instrument utilizing electrical biosensors-produces a standardized inoculum equivalent to a 0.5 McFarland directly from positive blood cultures. This proof-of-concept study demonstrates that eQUANT inocula prepared from clinically significant species of Enterobacterales were comparable to 0.5 McF inocula generated from bacterial colonies in both CFU/ml concentration and performance in antimicrobial susceptibility testing, with ≥ 95% essential and categorical agreement for VITEK2 and disk diffusion. The eQUANT, combined with a rapid, direct from positive blood culture identification technique, can allow the clinical laboratory to begin antimicrobial susceptibility testing using a standardized inoculum approximately 2-3 h after a blood culture flags positive. This has the potential to improve clinical practice by accelerating conventional antimicrobial susceptibility testing and the resulting targeted antibiotic therapy.

Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin.

Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKIIδ in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKIIδ also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKIIδ revealed ten phosphorylation sites, five of which are located within C2-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca2+ affinity of the recombinant C2F domain 10-fold, and increases the Ca2+ affinity of the C2C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKIIδ in IHCs.

A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Conformation of peptides bound to the transporter associated with antigen processing (TAP).

The ATP-binding cassette transporter associated with antigen processing (TAP) plays a key role in the adaptive immune defense against infected or malignantly transformed cells by translocating proteasomal degradation products into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. The broad substrate spectrum of TAP, rendering peptides from 8 to 40 residues, including even branched or modified molecules, suggests an unforeseen structural flexibility of the substrate-binding pocket. Here we used EPR spectroscopy to reveal conformational details of the bound peptides. Side-chain dynamics and environmental polarity were derived from covalently attached 2,2,5,5-tetramethylpyrrolidine-1-oxyl spin probes, whereas 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled peptides were used to detect backbone properties. Dependent on the spin probe's position, striking differences in affinity, dynamics, and polarity were found. The side-chains' mobility was strongly restricted at the ends of the peptide, whereas the central region was flexible, suggesting a central peptide bulge. In the end, double electron electron resonance allowed the determination of intrapeptide distances in doubly labeled peptides bound to TAP. Simulations based on a rotamer library led to the conclusion that peptides bind to TAP in an extended kinked structure, analogous to those bound to MHC class I proteins.

Single residue within the antigen translocation complex TAP controls the epitope repertoire by stabilizing a receptive conformation.

The recognition of virus infected or malignantly transformed cells by cytotoxic T lymphocytes critically depends on the transporter associated with antigen processing (TAP), which delivers proteasomal degradation products into the endoplasmic reticulum lumen for subsequent loading of major histocompatibility complex class I molecules. Here we have identified a single cysteinyl residue in the TAP complex that modulates peptide binding and translocation, thereby restricting the epitope repertoire. Cysteine 213 in human TAP2 was found to be part of a newly uncovered substrate-binding site crucial for peptide recognition. This residue contacts the peptide in the binding pocket in an orientated manner. The translocation complex can be reversibly inactivated by thiol modification of this cysteinyl residue. As part of an unexpected mechanism, this residue is crucial in complementing the binding pocket for a given subset of epitopes as well as in maintaining a substrate-receptive conformation of the translocation complex.

Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis.

The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.

Intracellular peptide transporters in human--compartmentalization of the "peptidome".

In the human genome, the five adenosine triphosphate (ATP)-binding cassette (ABC) half transporters ABCB2 (TAP1), ABCB3 (TAP2), ABCB9 (TAP-like), and in part, also ABCB8 and ABCB10 are closely related with regard to their structural and functional properties. Although targeted to different cellular compartments such as the endoplasmic reticulum (ER), lysosomes, and mitochondria, they are involved in intracellular peptide trafficking across membranes. The transporter associated with antigen processing (TAP1 and TAP2) constitute a key machinery in the major histocompatibility complex (MHC) class I-mediated cellular immune defense against infected or malignantly transformed cells. TAP translocates the cellular "peptidome" derived primarily from cytosolic proteasomal degradation into the ER lumen for presentation by MHC class I molecules. The homodimeric ABCB9 (TAP-like) complex located in lysosomal compartments shares structural and functional similarities to TAP; however, its biological role seems to be different from the MHC I antigen processing. ABCB8 and ABCB10 are targeted to the inner mitochondrial membrane. MDL1, the yeast homologue of ABCB10, is involved in the export of peptides derived from proteolysis of inner-membrane proteins into the intermembrane space. As such peptides are presented as minor histocompatibility antigens on the surface of mammalian cells, a physiological role of ABCB10 in the antigen processing can be accounted.

Mechanism of substrate sensing and signal transmission within an ABC transporter: use of a Trojan horse strategy.

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.

Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery.

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.