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Journal of Bacteriology, March 2008, p. 2249-2251, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01824-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Chaperone IpgC Copurifies with the Virulence Regulator MxiE{triangledown}

M. Carolina Pilonieta and George P. Munson*

Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida

Received 19 November 2007/ Accepted 31 December 2007


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ABSTRACT
 
The expression of a subset of Shigella flexneri virulence genes is dependent upon a cytoplasmic chaperone, IpgC, and an activator from the AraC/XylS family, MxiE. In this paper, we report that the chaperone forms a specific and stable heteromer with MxiE.


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TEXT
 
Shigella flexneri is the causative agent of bacillary dysentery, which is endemic throughout the world (6). S. flexneri has a type III secretion system for the delivery of proteins needed to colonize and invade the colonic epithelium (4, 6). Although many virulence genes are transcribed prior to secretion, the transcription of others is linked to the initiation of secretion. This latter group includes genes within the MxiE regulon, such as ipaH9.8, ospE2, ospC1, ospF, and virA (5, 7, 8). MxiE is a 29-kDa protein and a member of the AraC/XylS family of transcriptional regulators (3); however, its activity requires the 18-kDa protein IpgC, which functions as a coactivator for the transcription of virulence genes within the MxiE regulon (5, 7, 8). Prior to the secretion of invasion and effector proteins, IpgC is unable to serve as a coactivator with MxiE because, as a chaperone, it is sequestered in complexes with IpaB and IpaC (1, 9). Upon the secretion of IpaB and IpaC, however, IpgC is available to take on its subsequent role as a coactivator with MxiE (7, 9). Other pathogens, such as Chromobacterium violaceum, enterohemorrhagic Escherichia coli, Salmonella enterica serovar Typhimurium, and Yersinia enterocolitica, are likely to utilize similar regulatory systems, based upon homologies to MxiE and IpgC and, in some cases, experimental evidence (2, 7, 10, 17).

Unlike IpaB and IpaC, the stable expression of MxiE is not dependent upon IpgC. Nevertheless, it has been proposed that IpgC forms a heteromer with MxiE, as it does with IpaB and IpaC, and that the formation of a MxiE-IpgC complex is essential for the activation of virulence genes within the MxiE regulon (7, 13). However, the available experimental evidence challenges the validity of this model. With IpgC as the bait in a yeast two-hybrid system, there was no evidence of an interaction between MxiE and IpgC (7). MxiE also failed to copurify with His6-IpgC when it was expressed in S. flexneri or when the two proteins were coexpressed in E. coli under conditions that allowed for the copurification of either IpaB or IpaC with His6-IpgC (7, 12). Perhaps genetic and biochemical methods have failed to detect an MxiE-IpgC heteromer because it is short-lived, as has previously been suggested (7). However, this seems unlikely because it has been shown that the MxiE homolog InvF forms a stable complex with the IpgC homolog SicA from S. enterica serovar Typhimurium (2). A better understanding of the actual differences and similarities between these homologous systems would enhance future mechanistic studies by facilitating appropriate experimental design.

To elucidate the mechanism of MxiE- and IpgC-dependent transcription, we sought to develop an in vitro system to study the DNA binding of MxiE as a function of IpgC. AraC/XylS family members are notoriously insoluble in vitro (3, 16); therefore, we constructed a recombinant plasmid, pSFUM138, which expresses a fusion between maltose-binding protein (MBP) and MxiE from an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible tac promoter. The addition of MBP to the amino termini of other AraC/XylS family members has been shown to substantially enhance their solubility in vitro without perturbing their activity (11, 15). We also constructed a second plasmid, pSFUM139, which expresses MBP-MxiE in cis with His6-IpgC from tacp. The addition of a hexahistidyl epitope tag to IpgC has no effect on the activity of the protein (7, 12).

E. coli K-12 strains TB1/pSFUM138 (malE::mxiE) (TB1 was obtained from New England Biolabs) and TB1/pSFUM139 (malE::mxiE His6::ipgC) were cultured aerobically at 37°C in Luria-Bertani medium with ampicillin at 100 µg/ml and 0.2% (wt/vol) glucose until the optical absorbance of the culture reached 0.6 at 600 nm. The temperature was then reduced to 30°C, and the expression was induced by the addition of IPTG to a final concentration of 0.5 mM. Bacterial cells were harvested by centrifugation after 3 hours of induction and then resuspended in 10 ml of ice-cold lysis buffer (10 mM Tris-Cl [pH 7.6 at room temperature], 200 mM NaCl, 1 mM EDTA, 0.5 mM CaCl2, 5 mM β-mercaptoethanol, 100 µg/ml DNase I, 500 µg/ml lysozyme, 10% [vol/vol] glycerol). All subsequent purification steps were conducted at 4°C. Cells were lysed by passage through a French press, and the lysate was clarified by high-speed centrifugation. MBP-MxiE was then bound to an amylose column (New England Biolabs) equilibrated with buffer A (10 mM Tris-Cl [pH 7.6 at room temperature], 200 mM NaCl, 5 mM β-mercaptoethanol, 10% [vol/vol] glycerol) and eluted with buffer B (buffer A with 10 mM maltose). This procedure resulted in substantial purification of MBP-MxiE from both strains (Fig. 1). We also observed a protein that copurified with MBP-MxiE from strain TB1/pSFUM139 (malE::mxiE His6::ipgC) (Fig. 1, lane 4) but not when MBP-MxiE was purified from TB1/pSFUM138 (malE::mxiE) (Fig. 1, lane 2). The molecular mass of the copurifying protein was near the expected 19 kDa of His6-IpgC. Based on a previous report that IpgC and MxiE do not copurify (7), we did not anticipate these results, but subsequent Western blot analyses using an antibody (Cell Signaling Technology) that recognizes the hexahistidyl epitope tag confirmed that the 19-kDa protein is His6-IpgC (data not shown). The copurification of His6-IpgC with MBP-MxiE is the result of a specific interaction between the regulator and chaperone because His6-IpgC does not copurify with MBP (Fig. 2 and data not shown).


Figure 1
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  		FIG. 1. Copurification of His6-IpgC with MBP-MxiE. Coomassie blue-stained proteins that were expressed in strains TB1/pSFUM138 (malE::mxiE) or TB1/pSFUM139 (malE::mxiE his6::ipgC) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following amylose affinity chromatography. Lanes: 1 and 3, soluble proteins that were not retained on the amylose column; 2 and 4, soluble proteins in the column eluate. MBP-MxiE and His6-IpgC have expected molecular masses of 72 and 19 kDa, respectively. Std., protein mass standards.


Figure 2
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  		FIG. 2. IpgC does not bind MBP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Coomassie blue-stained proteins from strain TB1/pMalMxiEIpgC [malE::mxiE(44 to 251) ipgC] purified by amylose affinity and anion-exchange chromatography. Lanes: 1, eluate from the amylose column; 2, proteins not retained on the anion-exchange column; 3 to 6, eluate from the anion-exchange column. MBP-MxiE(44-251), MBP, and IpgC have expected molecular masses of 67, 43, and 18 kDa, respectively. Std., protein mass standards.

MxiE is encoded by two overlapping open reading frames that are out of frame with each other by –1 nucleotide. During transcription, an extra nucleotide is occasionally inserted into the nascent mRNA, resulting in a continuous mxiE open reading frame (14). The second open reading frame contains mxiE codons 44 to 251. Although MxiE(44-251) cannot activate transcription (14), we repeated our copurification protocol with MBP-MxiE(44-251) to determine if IpgC forms a complex with the truncated protein. MBP-MxiE(44-251) was expressed in cis with IpgC in strain TB1/pMalMxiEIpgC [malE::mxiE(44-251) ipgC] and purified on an amylose column, as described above. Analysis of the eluate revealed that IpgC copurified with MBP-MxiE(44-251) (Fig. 2, lane 1). Furthermore, copurification occurred in the absence of a hexahistidyl tag on IpgC. The eluate also contained a significant amount of MBP as the result of proteolytic cleavage of the MBP fusion protein during purification, presumably within the flexible linker between MBP and MxiE(44-251). The amylose column eluate was diluted fourfold with a dilution buffer (10 mM Tris-Cl [pH 7.6 at room temperature], 1 mM EDTA, 10 mM β-mercaptoethanol, 15% [vol/vol] glycerol) and then applied to an anion-exchange column (HiTrap Q FF; GE Healthcare) that had been equilibrated with Q buffer (dilution buffer with 50 mM NaCl). Afterward, the bound proteins were eluted from the column with a continuous gradient of 50 to 500 mM NaCl. Analysis of selected fractions revealed that both MBP-MxiE(44-251) and IpgC passed through the anion-exchange column without retention (Fig. 2, lane 2). In contrast, MBP bound to the column and was not eluted until the concentration of NaCl exceeded 90 mM (Fig. 2, lanes 3 to 5). These results are consistent with our previous conclusion that IpgC forms a specific complex with MxiE because IpgC can be separated from MBP by anion-exchange chromatography.

In our experience, the MxiE-IpgC complex is not transient. Rather, the heteromer is stable from approximately 15 mM to 1 M NaCl and resistant to the nonionic detergent Tween 20 at 0.25% (vol/vol). This is curious because previous attempts to copurify IpgC with MxiE from E. coli lysates have been unsuccessful (7). One clear methodological difference between this study and the previous work is that we used an amylose affinity column to copurify IpgC with MBP-MxiE, while the previous study used a metal ion affinity resin in an attempt to copurify His6-IpgC with MxiE. However, it is unlikely that this difference accounts for the different experimental outcomes, because SicA was copurified with InvF-His6 on a nickel agarose column (2). Perhaps when His6-IpgC is in a macromolecular complex with MxiE, its hexahistidyl tag is buried and unable to interact with metal ions. Nevertheless, the results of this study demonstrate that MxiE and IpgC form a stable heteromer, as do InvF and SicA (2). This also indicates that the two systems are likely to activate transcription via a similar mechanism. Although many remaining questions must be addressed before this mechanism is fully understood, it is probable that the formation of a long-lived MxiE-IpgC or InvF-SicA complex is an essential early step.


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ACKNOWLEDGMENTS
 
We thank A. T. Maurelli and S. M. Payne for providing the strains of S. flexneri.

This work was supported by Public Health Service award AI 057648 from the National Institutes of Health and the University of Miami Miller School of Medicine.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Miami Miller School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101. Phone: (305) 243-5317. Fax: (305) 243-4623. E-mail: gmunson{at}miami.edu Back

{triangledown} Published ahead of print on 11 January 2008. Back


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Journal of Bacteriology, March 2008, p. 2249-2251, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01824-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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