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PNAS 109 (38): 15461-15466

Copyright © 2012 by the National Academy of Sciences.

Protein cysteine phosphorylation of SarA/MgrA family transcriptional regulators mediates bacterial virulence and antibiotic resistance

Fei Suna, Yue Dingb, Quanjiang Jia, Zhongjie Liangb, Xin Denga, Catherine C. L. Wongc, Chengqi Yia, Liang Zhanga, Sherrie Xiea, Sophie Alvarezd, Leslie M. Hicksd, Cheng Luob, Hualiang Jiangb, Lefu Lanb,1, and Chuan Hea,1

aDepartment of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; bShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; c The Scripps Research Institute, San Diego, CA 92121; and d Donald Danforth Plant Science Center, St. Louis, MO 63132


Figure 01
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Fig. 1.. S. aureus SarA/MgrA family protein. (A) Model of gene regulation by the oxidation-sensing SarA/MgrA/SarZ. The N-terminal Cys residue, highly conserved in SarA/MgrA/SarZ, is subject to oxidation by ROSs, thus leading to dissociation from DNA. Phosphorylation of the same Cys residue mediated by Stk1-Stp1 might also modulate their target gene regulation. (B) Sequence alignment of SarZ, MgrA, and SarA. The Cys residue (indicated by an arrow) and the surrounding conserved residues are highlighted in red. The alignment was performed with ClustalW2 (61).

 

Figure 02
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Fig. 2.. Phosphorylation of SarA family proteins (SarA, MgrA, and SarZ). (A) The Cys-to-Ser substitution abolished the phosphorylation of SarA, MgrA, and SarZ. WT, cell extract from the wild-type strain Newman; {Delta}stp1, cell extract from {Delta}stp1 strain (an in-frame deletion mutant of stp1). Protein bands stained with Coomassie blue after autoradiography were shown. (B) The phosphatase Stp1 dephosphorylates phospho-SarA (lane 3). The phospho-SarA was treated with Stp1 at 37 °C for 10 min before analysis. (C) The phosphorylation activity of cell extract from {Delta}stp1 was restored by complementation with pYJ335::stp1 (lane 3). {Delta}stp1-C, {Delta}stp1/pYJ335::stp1. (D) Overexpression of Stk1 enhanced phosphorylation of SarA (lane 3). stk1++, cell extract from the wild-type strain carrying pYJ335::stk1, in which the expression of stk1 was induced by anhydrotetracycline (1 μg/mL). (E) Deactivation of stk1 abolished phosphorylation of SarA (lane 3). stp1-I, cell extract from the mutant with bursa aurealis transposon insertion in stp1 that deactivates both stp1 and stk1.

 

Figure 03
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Fig. 3.. LC-MS/MS identification of Cys-phosphorylation of SarA and MgrA. (A) LC-MS/MS spectrum of the phosphopeptide I6NpCFELLSMVTYADKLK23 (observed m/z 2182.0140 Da corresponding to apo-peptide theoretical mass of 2102.0393 Da + 1 phosphate group 79.9747 Da) obtained after trypsin digestion of phospho-SarA. The b5 and b6 fragment ions corresponding to I6NDpCF and I6NDpCFE, respectively (observed m/z 673.2051 and 802.2247 corresponding to apo-fragment + 1 phosphate group 79.9437 Da) indicates the presence of phospho-Cys rather than on Ser or Thr. The phospho-Cys-9-Phe-10 (pCF) fragment is also highlighted by the mass difference of b5 and b3 fragment ions. (B) LC-MS/MS spectrum of the phosphopeptide E9QLpCFSLYNAQR20 (observed m/z 1550.6555 Da corresponding to apo-peptide experimental mass of 1470.6967 Da + 1 phosphate group 79.9588 Da) obtained after trypsin digestion of phospho-MgrA. The characteristic mass difference of the phospho-Cys-12 is highlighted. Fragment ions y5 and y7 indicate that phosphorylation is not on Ser-14 or Thr-16 and fragment y9 shows a mass shift of 80 Da from the phosphorylation of Cys-12. Individual fragments are labeled based on the b- or y-ion nomenclature. The phospho-fragments are colored red. Fragment ions arising from the neutral loss of water (–18 Da) are marked with a zero (0) and fragment ions with the loss of ammonia (–17 Da) are marked with an asterisk (*).

 

Figure 04
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Fig. 4.. Cys-phosphorylation modulates bacterial virulence and antibiotic resistance. (A) Western blot analysis of phospho-SarA from cell extract. SarA, His6-SarA enriched with Ni-NTA beads from whole-cell extract of {Delta}stp1-sarA/pYJ335::sarA; SarAC9S, His6-SarAC9S from {Delta}stp1-sarA/pYJ335::sarAC9S; Control, the recombinant SarA expressed by E. coli after in vitro phosphorylation as a positive control. Phospho-Ab, antiphospho-Thr antibody; His-tag, InVision His-tag in-gel stain (Invitrogen). {Delta}stp1-sarA, a double mutant with in-frame, unmarked stp1 deletion and bursa aurealis transposon insertion in sarA. (B) Hemolysis on the sheep blood agar. The strains tested were spotted on 5% (vol/vol) sheep blood agar plate. Zones of clearance indicate hemolysis. (C) Western blot analysis of the production of α-hemolysin (Hla) (see SI Experimental Procedures). (D) Vancomycin resistance assays. Aliquots (10 μL) of the diluted overnight cultures for each strain (5 x 106 CFU/mL) were spotted onto the tryptic soy agar (TSA) plates without (Upper) or with 1.6 μg/mL vancomycin (Lower). Both plates were supplemented with 1 μg/mL anhydrotetracycline (aTc) to induce the expression of SarA. (E and F) Effect of mutation of stp1 on the virulence of S. aureus in a mouse model of abscess formation. The wild-type strain Newman and the stp1 deletion mutant ({Delta}stp1) were used to infect 10 mice each via retro-orbital injection. After 5 d, S. aureus colonization in murine liver (E) or kidney (F) was measured. Each circle represents one mouse. The horizontal black line represents the mean log10 CFU on the y axis. The statistical difference between mutant and wild-type strains was determined by Student t test (two-tailed). The limit of detection for organ infection is 100 CFU per organ.

 


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