Emergence of Ceftazidime-Avibactam Resistance Due to Plasmid-Borne blaKPC-3 Mutations during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

Ceftazidime-avibactam is a novel ß-lactam/ß-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne blaKPC-3, which were not present in baseline isolates. blaKPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and blaKPC cloning into competent Escherichia coli In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ≥4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced ≥4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of blaKPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to blaKPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring blaKPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.

Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection.

A recently described fluorescence biosensor platform utilizes single-chain Fv (scFvs) that selectively bind and activate fluorogen molecules. In this report we investigated the display of tandem scFv biosensors at the surface of mammalian cells with the aim of advancing current fluorescence detection strategies. We initially screened different peptide linkers to separate each scFv unit, and discovered that tandem proteins joined by either flexible or α-helical linkers properly fold and display at the surface of mammalian cells. Accordingly, we performed a combinatorial scFv-dimer study and identified that fluorescence activation correlated with the cellular location (membrane distal versus proximal) and selections of the different scFvs. Furthermore, in vitro measurements showed that the stability of each scFv monomer unit influenced the folding and cell surface activities of tandem scFvs. Additionally, we investigated the absence or poor signals from some scFv-dimer combinations and discovered that intramolecular and intermolecular scFv chain mispairings led to protein misfolding and/or secretory-pathway-mediated degradation. Furthermore, when tandem scFvs were utilized as fluorescence reporter tags with surface receptors, the biosensor unit and target protein showed independent activities. Thus, the live cell application of tandem scFvs permitted advanced detection of target proteins via fluorescence signal amplification, Förster resonance energy transfer resulting in the increase of Stokes shift and multi-color vesicular traffic of surface receptors.

A single-chain-variable-fragment fluorescence biosensor activates fluorogens from dissimilar chemical families.

Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.

Hsp70 and Hsp90 multichaperone complexes sequentially regulate thiazide-sensitive cotransporter endoplasmic reticulum-associated degradation and biogenesis.

The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC. Analyses of NCC immunoprecipitates revealed that the cotransporter formed complexes with the core chaperones Hsp90, Hsp70, and Hsp40. Disruption of Hsp90 function accelerated NCC degradation, suggesting that Hsp90 promotes NCC folding. In addition, two cochaperones, the C terminus of Hsp70-interacting protein (CHIP) and the Hsp70/Hsp90 organizer protein, were associated with NCC. Although CHIP, an E3 ubiquitin ligase, promoted NCC ubiquitination and ERAD, the Hsp70/Hsp90 organizer protein stabilized NCC turnover, indicating that these two proteins differentially remodel the core chaperone systems to favor cotransporter degradation and biogenesis, respectively. Adjusting the folding environment in mammalian cells via reduced temperature enhanced NCC biosynthetic trafficking, increased Hsp90-NCC interaction, and diminished binding to Hsp70. In contrast, cotransporters harboring disease-causing mutations that impair NCC biogenesis failed to escape ERAD as efficiently as the wild type protein when cells were incubated at a lower temperature. Instead, these mutants interacted more strongly with Hsp70, Hsp40, and CHIP, consistent with a role for the Hsp70/Hsp40 system in selecting misfolded NCC for ERAD. Collectively, these observations indicate that Hsp70 and Hsp90 comprise two functionally distinct ER quality control checkpoints that sequentially monitor NCC biogenesis.

The thiazide-sensitive NaCl cotransporter is targeted for chaperone-dependent endoplasmic reticulum-associated degradation.

The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.

Half-site inhibition of dimeric kinesin head domains by monomeric tail domains.

The two heavy chains of kinesin-1 are dimerized through extensive coiled coil regions and fold into an inactive conformation through interaction of the C-terminal tail domains with the N-terminal motor (head) domains. Although this potentially allows a dimer of tail domains to interact symmetrically with a dimer of head domains, we report here that only one of the two available monomeric tail peptides is sufficient for tight binding and inhibition of a dimer of head domains. With a dimeric tail construct, the other tail peptide does not make tight contact with the head dimer and can bind a second head dimer to form a complex containing one tail dimer and two head dimers. The IAK domain and neighboring positively charged region of the tail is sufficient for tight half-site interaction with a dimer of heads. The interaction of tails with monomeric heads is weak, but a head dimer produced by the dimerization of the neck coil is not required because an artificial dimer of head domains also binds monomeric tail peptides with half-site stoichiometry in the complete absence of the native neck coil. The binding of tail peptides to head dimers is fast and readily reversible as determined by FRET between mant-ADP bound to the head dimer and a tail labeled with GFP. The association and dissociation rates are 81 microM(-1) s(-1) and 32 s(-1), respectively. This half-site interaction suggests that the second tail peptide in a folded kinesin-1 might be available to bind other molecules while kinesin-1 remained folded.