CydDC functions as a cytoplasmic cystine reductase to sensitize Escherichia coli to oxidative stress and aminoglycosides Alexander Mironova,b, Tatyana Sereginaa,b, Konstantin Shatalinc, Maxim Nagornykha, Rustem Shakulova, and Evgeny Nudlerc,d,1 aDepartment of Molecular Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Science, 119991 Moscow, Russia; bCenter for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; cDepartment of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; and dHHMI, New York University School of Medicine, New York, NY 10016 Edited by James J. Collins, Massachusetts Institute of Technology, Boston, MA, and approved August 6, 2020 (received for review April 22, 2020) L-cysteine is the source of all bacterial sulfurous biomolecules. dependent manner (10, 13, 14). However, whether CydDC is However, the cytoplasmic level of L-cysteine must be tightly reg- involved in exporting L-cysteine or GSH to the periplasm of living ulated due to its propensity to reduce iron and drive damaging cells remains unknown, as is the mechanism by which this complex Fenton chemistry. It has been proposed that in Escherichia coli the maintains the cellular redox status. component of cytochrome bd-I terminal oxidase, the CydDC com- Because of its highly labile nature, dynamic distribution be- plex, shuttles excessive L-cysteine from the cytoplasm to the peri- tween cellular compartments, and overall low concentration, direct plasm, thereby maintaining redox homeostasis. Here, we provide and accurate measurement of intracellular L-cysteine has been evidence for an alternative function of CydDC by demonstrating highly challenging, leading to inconclusive and sometime contra- that the cydD phenotype, unlike that of the bona fide L-cysteine eamA tcyP. dictory results (3, 8). Recently, we demonstrated that the accumu- exporter , parallels that of the L-cystine importer Chro- E. coli mosomal induction of eamA, but not of cydDC, from a strong lation of L-cysteine in directly correlates with the level of enzymatically produced H2S (15). The sequential action of cyto- pLtetO-1 promoter (Ptet) leads to the increased level of extracellu- BIOCHEMISTRY lar L-cysteine, whereas induction of cydDC or tcyP causes the ac- plasmic aspartate aminotransferase (AspC) and 3-mercaptopyruvate cumulation of cytoplasmic L-cysteine. Congruently, inactivation of sulfurtransferase (3MST) accounts for most H2Sproductionin cydD renders cells resistant to hydrogen peroxide and to amino- E. coli grown in rich media (15). Cytoplasmic AspC and 3MST, glycoside antibiotics. In contrast, induction of cydDC sensitizes which are constitutively expressed, utilize L-cysteine as their pri- cells to oxidative stress and aminoglycosides, which can be sup- mary substrate, helping to maintain its concentration at nontoxic pressed by eamA overexpression. Furthermore, inactivation of the levels under normal growth and stress conditions (15–17). Volatile fur) cydDC tcyP ferric uptake regulator ( in Ptet- or Ptet- cells results and freely diffusible 3MST-derived H2S can be readily detected in in dramatic loss of survival, whereas catalase (katG) overexpres- a growing culture. We used this quantitative measurement of in- sion suppresses the hypersensitivity of both strains to H2O2. tracellular L-cysteine to study the role of CydDC in the L-cysteine/ These results establish CydDC as a reducer of cytoplasmic cys- cystine shuttle system and its impact on cellular resistance to tine, as opposed to an L-cysteine exporter, and further elucidate a oxidative stress and antibiotics. link between oxidative stress, antibiotic resistance, and sulfur metabolism. Significance bacteria | oxidative stress | L-cysteine | aminoglycosides | cytochrome The CydDC complex is involved in the assembly of cytochrome bd-I, a terminal oxidase of the respiratory chain required for -cysteine is an essential building block and source of redox- growth under low oxygen conditions. It has been suggested Lactive sulfhydryl groups for proteins, glutathione (GSH), and that CydDC shuttles excessive L-cysteine from the cytoplasm to many other biomolecules. Yet, L-cysteine is also toxic because of the periplasm, thereby maintaining bacterial redox homeosta- its ability to promote the Fenton reaction and generate damag- sis. Here, we demonstrate that the principle function of CydDC ing hydroxyl radicals (1–6). Therefore, bacterial cells maintain is in maintaining the reduced state of cytoplasmic L-cysteine, as the low level of intracellular L-cysteine by coordinating its bio- opposed to exporting L-cysteine, thereby providing an impor- synthesis, utilization, oxidation, and transport (7). Escherichia coli tcyP tcyJ, tant connection between sulfur metabolism, oxidative stress, Two L-cysteine/cystine importers, and and resistance to antibiotics. In particular, we establish the function synchronously with the L-cysteine exporter EamA (8) to critical role of cytoplasmic L-cysteine in mediating the toxicity constitute the L-cysteine/cystine shuttle system, which plays an of aminoglycosides. Thus, understanding the mechanisms that important role in oxidative stress tolerance by providing reducing control the bacterial redox state can lead to more effective equivalents to the periplasm (9). It has also been suggested that a strategies to counter bacterial resistance and tolerance. putative ABC-type transporter, CydDC, a heterodimeric compo- bd nent of cytochrome -I terminal oxidase, is involved in L-cysteine Author contributions: E.N. designed research; A.M., T.S., K.S., and M.N. performed re- efflux from the cytoplasm to the periplasm (10). Cells deficient in search; R.S. contributed new reagents/analytic tools; A.M., T.S., M.N., and R.S. analyzed cydD or cydC exhibit hypersensitivity to high concentrations of data; and E.N. wrote the paper. L-cysteine and DTT (10, 11), suggesting that CydDC could be The authors declare no competing interest. involved in periplasmic sulfhydryl homeostasis. Additional phe- This article is a PNAS Direct Submission. notypes of cydDC include temperature sensitivity and stationary Published under the PNAS license. phase arrest, which can be suppressed by exogenous reductants 1To whom correspondence may be addressed. Email: [email protected]. (12). The conclusion that CydDC exports L-cysteine and GSH to This article contains supporting information online at https://www.pnas.org/lookup/suppl/ the periplasm was based on the observation that everted membrane doi:10.1073/pnas.2007817117/-/DCSupplemental. vesicles uptake L-cysteine or GSH in an ATP- and CydDC- www.pnas.org/cgi/doi/10.1073/pnas.2007817117 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 Results and Discussion Ptet-mstA (SI Appendix,Fig.S1). The level of H2SinΔcydD cells -cydDC H S Production as a Function of the L-Cysteine/Cystine Shuttle System. was approximately the same as in the control, whereas Ptet 2 B To establish the relationship between the E. coli L-cysteine/cystine cells generated more H2S (Fig. 1 ), arguing that hyperactive L shuttle system and H2SproductionweutilizedH2S-deficient cells CydDC increases, and does not decrease, the level of -cysteine in (ΔmstA) and cells carrying a chromosomal copy of mstA under a the cytoplasm. Indeed, CydDC and EamA demonstrated the op- A B strong pLtetO-1 promoter (P -mstA) that generate ∼2.5-fold posite effects on H2S production (compare Fig. 1 and ). tet -cydDC ΔeamA more H S than do wild-type (WT) cells (15) (Fig. 1A). These Moreover, a combination of Ptet with further in- 2 ΔeamA -cydDC reference strains were used as the genetic backgrounds for the creased H2Sproductionascomparedto or Ptet B construction of chromosomal deletions and Ptet versions of the alone (Fig. 1 ). principle L-cysteine/cystine shuttle genes: tcyP and eamA (SI Ap- It has been noted that the overexpression of CydDC from a pendix,TableS1). Deletion of the L-cystine importer, tcyP,low- high-copy-number plasmid does not result in the accumulation of ered H2SinWTandPtet-mstA cells, whereas the inactivation of L-cysteine in the culture medium (18), contrary to what is expected the L-cysteine exporter eamA increases H2SinWTandPtet-mstA of a putative L-cysteine exporter. Accordingly, we also failed to - cells (Fig. 1A). The contrasting effects of tcyP and eamA on H2S detect any increase of L-cysteine in the culture medium of Ptet production became even more apparent when the two genes were cydDC cells, as compared to WT cells (Fig. 1C). In contrast, cells eamA overexpressed. Ptet-eamA cells exhibited a drastic drop in H2S overexpressing EamA (Ptet- ) significantly increased the production, whereas Ptet-tcyP cells produced substantially more H2S L-cysteine concentration in the growth medium. These data argue as compared to control cells (Fig. 1A). These results demonstrate that CydDC does not export reduced thiols from the cytoplasm. that the level of MstA-derived H2S reflects the distribution of We hypothesized that CydDC elevates the level of cytoplasmic L-cysteine/cystine between the cytoplasm and periplasm and can be L-cysteine, and, hence, that of MstA-derived H2S, by stimulating used to quantitatively assess the cytoplasmic L-cysteine content. cytoplasmic cystine reduction. Indeed, deleting cydD in Ptet-tcyP cells, which overproduce the dedicated cystine importer, diminished CydDC Does Not Export L-Cysteine