Exploring Metalloproteome Remodeling in Calprotectin-Stressed <i>Acinetobacter baumannii</i> Using Chemoproteomics

The growth of bacterial pathogens is limited by nutritional immunity, where the infected host deploys transition metal scavenging proteins including calprotectin (CP) to starve the bacterium of essential transition metals. Prior work reveals that CP induces a significant Zn- and Fe-starvation response in the Gram-negative opportunistic pathogen <i>Acinetobacter baumannii</i> in liquid culture. Here, we employ a quantitative chemoproteomics platform to pinpoint changes in abundance-corrected cysteine reactivity─and by extension cellular metal occupancy in metalloenzymes─that occur when <i>A. baumannii</i> is challenged with physiological CP in liquid culture relative to an untreated WT control. Changes in protein abundance with CP stress reveal a pronounced Zn-limitation and Fe-starvation response and reciprocal regulation of three enzymes of central carbon metabolism, including aconitase. A majority of the 2645 quantifiable Cys-containing peptides that show an increase in abundance-corrected Cys reactivity (150) are derived from known Zn-, Fe-, and Fe-S-cluster proteins, revealing a significant decrease in metal occupancy (undermetalation) across the proteome. Myriad cell processes are impacted by undermetalation, including enzymes that function in the TCA cycle and respiration, GTP metabolism, ribosome remodeling, tRNA charging, and proteostasis. In an effort to identify an undemetalated client enzyme for the candidate GTPase-powered metallochaperone ZigA, we performed this chemoproteomics experiment in a CP-stressed Δ<i>zigA</i> strain relative the CP-stressed wild-type strain. These findings reveal that the loss of ZigA is effectively silent in this assay. We conclude that CP induces a widespread, negative impact on the metalation status of the metalloproteome that results in a significant nutrient limitation response.