CydDC functions as a cytoplasmic cystine reductase to sensitize Escherichia coli to oxidative stress and aminoglycosides

Abstract

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2. These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.

Whole-genome sequencing and gene sharing network analysis powered by machine learning identifies antibiotic resistance sharing between animals, humans and environment in livestock farming

“…Importantly, purine metabolism and synthesis pathways have been previously found to be enriched in multidrug resistant bacteria [ 22 ]. The transcription factor fur present in the cefotaxime, ampicillin, ampicillin/clavulanic acid and doxycycline models, represses genes involved in high affinity iron sequestration important in virulence [ 55 ] and is important in conferring protection from oxidative stress [ 56 ]. The gene tus , involved in the termination of DNA replication, was significant in doxycycline, ampicillin/sulbactam, minocycline and streptomycin.…”

The Inactivation of LPS Biosynthesis Genes in E. coli Cells Leads to Oxidative Stress

“…Cystine is present in bacterial cells in trace amounts that cannot be detected [ 33 ]. Currently, cysteine is considered one of the main protectors against ROS in the periplasm of gram-negative bacteria [ 34 , 35 ]. On the other hand, the concentration of cysteine in the cell cytoplasm is strictly controlled, since an excess of cysteine can promote the genotoxic Fenton reaction [ 35 , 36 ].…”

“…Currently, cysteine is considered one of the main protectors against ROS in the periplasm of gram-negative bacteria [ 34 , 35 ]. On the other hand, the concentration of cysteine in the cell cytoplasm is strictly controlled, since an excess of cysteine can promote the genotoxic Fenton reaction [ 35 , 36 ]. As a reason for the increased sensitivity of mutants to antibiotics, one can observe a decrease in the intracellular concentration of cysteine ( Figure 7 ).…”

Dissecting the conformational complexity and flipping mechanism of a prokaryotic heme transporter

“…We next tested affinity-purified CydDC for ATPase activity in the presence of various potential substrates, primarily using the Malachite green phosphate assay (Supplementary Fig. 1) ( 29, 30, 32, 33 ). In agreement with our growth complementation data, but in contrast to previous reports, we found that GSH and L-Cys (and their oxidized counterparts GSSG and CSSC) failed to induce hydrolysis, whereas heme stimulated the ATPase activity of CydDC in a concentration-dependent manner (Fig.…”

“…This observation motivated the assumption that heme represents a prosthetic group of a periplasmic redox sensor domain for regulation of transporter activity ( 17, 22 ). A recent work fundamentally challenged the transporter function of CydDC and proposed that it operates as a membrane-bound enzyme catalyzing the reduction of cytoplasmic cystine (CSSC) to L-Cys ( 29 ).…”