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BackgroundCefazolin is becoming first-line therapy for MSSA infections since it appears to be better tolerated than isoxazolyl penicillins with similar outcomes. An important concern when using cephalosporins as first-line therapy for MSSA is the CzIE, defined as MICs ≥ 16 µg/mL when performed at high bacterial inoculum (~107 CFU/mL) compared with standard inoculum (~105 CFU/mL). We postulated that release of BlaZ (a lipoprotein) to the extracellular milieu is the mechanism responsible for the CzIE. Confirmation of this phenomenon would permit developing a rapid test to identify this phenomenon in clinical settings.MethodsWe monitored the hydrolysis of 50 μM of nitrocefin by S. aureus supernatants after induction with ampicillin (150 µg/mL) for 1 h. A total of 150 μL of supernatants (after centrifugation) was incubated with 50 μM nitrocefin at 25°C in 20 mM HEPES, pH 7.4, 100 mM NaCl for 30 minutes. Nitrocefin hydrolysis was monitored by following the change in absorbance at 482 resulting from opening of the β-lactam ring of nitrocefin. Visual inspection to monitor color changes was also performed. We initially used 3 strains of MSSA, (i) S. aureus TX0117, a well-characterized strain that exhibits the CzIE; (ii) TX0117c, a derivative of TX0117 that harbors a mutation inactivating BlaZ and abolishing the CzIE, and (iii) ATCC 29213 a BlaZ-positive strain that lacks the CzIE. Subsequently, we validated the methodology in 10 South American isolates of different backgrounds that had been previously characterized for the CZIE.ResultsA statistically significant difference in ODs after 30 minutes was observed in TX0117 (CzIE) vs. TX0117c (no CzIE) and ATCC 29213 (no CzIE) (all P < 0.001), suggesting high BlaZ activity in supernatants of TX0117 and supporting the release of the enzyme as the main mechanism of the CzIE. All South American isolates that exhibited the CzIE were identified by the nitrocefin assay. Of note, isolates producing Type C BlaZ gave a weaker reaction, although still significantly different from isolates without the CzIE. Hydrolysis of nitrocefin was also readily detectable by visual inspection.ConclusionThe CzIE is likely due to release of BlaZ to the extracellular milieu. A rapid test that can readily identify MSSA strains exhibiting the CzIE is feasible.Disclosures W. Miller, Merck: Investigator, Research support. C. Arias, Merck & Co., Inc.: Grant Investigator, Research support; MeMed: Grant Investigator, Research support; Allergan: Grant Investigator, Research support.

In starving yeast exposed to thermal stress, a transient drop in intracellular pH helps to trigger the heat shock response.

Neurodegenerative disease affects millions of Americans every year, through diagnoses such as Alzheimer's, Parkinson's, and Huntington's diseases. One factor linked to formation of these aggregates is damage sustained to proteins by oxidative stress. Cellular protein homeostasis (proteostasis) relies on the ubiquitous Hsp70 chaperone family. Hsp70 activity has been previously shown to be modulated by modification of two key cysteines in the ATPase domain by oxidizing or thiol-modifying compounds. To investigate the biological consequences of cysteine modification on the Hsp70 Ssa1 in budding yeast, we generated cysteine null (cysteine to serine) and oxidomimetic (cysteine to aspartic acid) mutant variants of both C264 and C303 and demonstrate reduced ATP binding, hydrolysis and protein folding properties in both the oxidomimetic as well as hydrogen peroxide-treated Ssa1. In contrast, cysteine nullification rendered Ssa1 insensitive to oxidative inhibition. The oxidomimetic ssa1-2CD (C264D, C303D) allele was unable to function as the sole Ssa1 isoform in yeast cells and also exhibited dominant negative effects on cell growth and viability. Ssa1 binds to and represses Hsf1, the major transcription factor controlling the heat shock response, and the oxidomimetic Ssa1 failed to stably interact with Hsf1, resulting in constitutive activation of the heat shock response. Consistent with the in vitro findings, ssa1-2CD cells were compromised for de novo folding, post-stress protein refolding and in regulated degradation of a model terminally misfolded protein. Together these findings pinpoint Hsp70 as a key link between oxidative stress and proteostasis, information critical to understanding cytoprotective systems that prevent and manage cellular insults underlying complex disease states.