A Disulfide Bond in the Membrane Protein IgaA Is Essential for Repression of the RcsCDB System

Abstract

IgaA is an integral inner membrane protein that was discovered as repressor of the RcsCDB phosphorelay system in the intracellular pathogen Salmonella enterica serovar Typhimurium. The RcsCDB system, conserved in many members of the family Enterobacteriaceae, regulates expression of varied processes including motility, biofilm formation, virulence and response to envelope stress. IgaA is an essential protein to which, in response to envelope perturbation, the outer membrane lipoprotein RcsF has been proposed to bind in order to activate the RcsCDB phosphorelay. Envelope stress has also been reported to be sensed by a surface exposed domain of RcsF. These observations support a tight control of the RcsCDB system by RcsF and IgaA via mechanisms that, however, remain unknown. Interestingly, RcsF and IgaA have four conserved cysteine residues in loops exposed to the periplasmic space. Two non-consecutive disulfide bonds were shown to be required for RcsF function. Here, we report mutagenesis studies supporting the presence of one disulfide bond (C404-C425) in the major periplasmic loop of IgaA that is essential for repression of the RcsCDB phosphorelay. Our data therefore suggest that the redox state of the periplasm may be critical for the control of the RcsCDB system by its two upstream regulators, RcsF and IgaA.

Keywords: IgaA; RcsCDB; Salmonella; cysteine; disulfide bond; periplasmic domain.

FIGURE 1

The integral inner membrane protein IgaA has four conserved periplasmic cysteines. (A) Predicted topology of the Salmonella Typhimurium IgaA protein of 710 amino acids length. The periplasmic cysteines C404, C425, C498, and C504 as well as the cytosolic R188 residue important for IgaA function, are shown. (B) Alignment of the periplasmic domain of distinct IgaA orthologs from the indicated members of the Enterobacteriaceae family. Alignment was performed with the Clustal Omega tool.

FIGURE 2

Salmonella Typhimurium IgaA variants in conserved cysteines show distinct capacities to repress the RcsCDB system. (A) IgaA was detected in western assays using samples obtained from bacteria grown in LB medium at 37°C to mid-exponential (OD₆₀₀ = 0.2) or stationary (OD₆₀₀ = 2.0) phase. Note the degradation of the IgaA cysteine variants when bacteria reach the stationary phase. The previously characterized IgaA variant R188H (Dominguez-Bernal et al., 2004) was used as control of protein unstable in stationary phase. Numbers on the left indicate the position of molecular weight markers (in kDa). (B) Activity of the RcsCDB system monitored with a gmm::lacZ reporter fusion in bacteria growing to exponential or stationary phases. gmm encodes an enzyme involved in synthesis of the colanic acid capsule and is positively regulated by the RcsCDB system. Data are the average and standard deviation of three independent experiments. The averages are shown as numbers on top of the respective bars.

FIGURE 3

The periplasmic cysteines C404 and C425 are essential for the negative regulation that IgaA imposes over the RcsCDB phosphorelay. (A) Mucoid phenotype of strains producing the C404S, C425S, C504S, and R188H variants. (B) Motility test in soft agar. Note that the high repression of motility in the strain producing the C404S variant. A graphic showing motility halo diameter (average and standard deviation from three independent assays) is also depicted.

FIGURE 4

The periplasmic domain of IgaA has a C404–C425 disulfide bond essential for function that can be formed independently of the major disulfide oxidoreductase DsbA. (A) Alkylation assays with the 4′-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) reagent reveal electrophoretic shifts in wild-type IgaA but not in a variant lacking the periplasmic domain (DI358-W652). Note the co-existence of distinct forms in the IgaA-R188H mutant and the lack of effect of a ΔdsbA mutation in the presence of IgaA forms with distinct electrophoretic mobility. A scheme is shown denoting the changes expected for DTT/AMS incubations for an example of one disulfide bond. (B) AMS alkylation assays in the IgaA variants C404S, C425S, C498S, and C504S. Note that the lack of C404 results in no major changes in electrophoretic mobility in the samples treated or not with DTT. (C) Proposed configuration of the C404–C425 disulfide bond in the periplasmic domain of IgaA. (D) Detection of non-native disulfide bridges in the C404S–C498S and C404S–C504S IgaA variants. Note the similarity of electrophoretic mobility of the non-alkylated and alkylated samples not treated with DTT (see text for details).