Ceftazidime-avibactam is a novel ␤-lactam/␤-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidimeavibactam 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. Wholegenome 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 bla KPC-3 , which were not present in baseline isolates. bla KPC-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 bla KPC 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 Ͼ D179Y Ͼ V240G. Remarkably, mutations reduced meropenem MICs Ն4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced Ն4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of bla KPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to bla KPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring bla KPC-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.

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 µM−1s−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.

Background:The cation chloride cotransporter NCC is degraded by undefined mechanisms. Results: NCC requires specific conserved machinery for chaperone-dependent recognition, ubiquitination, and proteasomal routing. Conclusion: NCC exhibits distinct ERAD requirements, which correlate with its transmembrane topology and distinguish it from other clients. Significance: These ER quality control components process misfolded conformational intermediates of NCC and other structurally related cotransporters that are vital for human health.

Background: An incompletely defined system of cytoplasmic chaperones mediates thiazide-sensitive cotransporter (NCC) ER quality control. Results: Hsp70 and Hsp90 select NCC for cochaperone-regulated ERAD or biosynthetic maturation. Conclusion: Hsp70 and Hsp90 sequentially monitor early stages of NCC biogenesis. Significance: Differential interaction of aberrantly folded NCC with these chaperones likely contributes to the molecular basis of hereditary salt wasting and hypertension resistance.

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.