Delineation of the LcrV-interaction domain of LcrG

LcrG and LcrV physically interact in Y. pestis [4], and that interaction plays a significant role in the regulation of Yops secretion [20]. LcrG and LcrV interact in a yeast two-hybrid assay [20], therefore this technique was used to further investigate the region of LcrG required for its interaction with LcrV. Truncations of LcrG were constructed and fused to the GAL4 activation domain of pACT2. These plasmids were co-transformed into yeast strain Y187 with full-length LcrV fused to the GAL4 DNA binding domain of plasmid pAS2-1 to determine if the LcrG truncations could continue to participate in an interaction with LcrV. Using a filter-lift assay as a qualitative determination of an interaction and a liquid β-galactosidase assay to quantify the results, we found that the LcrV-interaction domain of LcrG lies in the N-terminus of the protein. The smallest linear region of LcrG that was still capable of interacting with LcrV was determined to be amino acids 7–40 (Fig. 1; shown highlighted in yellow in Fig. 2). Deletion of five additional amino acids from the N-terminus of the 7–40 construct or seven amino acids from the C-terminus of the 7–40 construct eliminated our ability to detect an interaction using this system (Fig. 1). Interestingly, this approximate region of LcrG (amino acids 7–35) is predicted to form a coiled-coil domain when LcrG is analyzed using the COILS algorithm [26] (data not shown). These results suggest that the LcrG-LcrV interaction requires the N-terminus of LcrG and may involve a coiled-coil domain.

Alanine-scanning mutagenesis of the LcrG N-terminus

'Radical' site-directed mutagenesis of LcrG allows the LcrG-LcrV interaction to be disrupted

The alanine at position 16 was previously identified as a critical residue in the LcrG-LcrV interaction [20]. Two mutations were characterized at this position (A16R and A16D) and they failed to interact with LcrV, showing that single substitutions of this kind can disrupt the LcrG-LcrV interaction [20]. Additional data from this study showed that a substitution of the leucine at position 30 with an arginine also prevented the LcrG-LcrV interaction in yeast (Table 2). Interestingly, when L30 is replaced with alanine (Table 1) or threonine (Table 2), the interaction is not effected. Mutagenesis of the serine at position 23 to arginine resulted in an LcrG variant that weakly interacted with LcrV as determined by a filter-lift assay for β-galactosidase activity. The color change of the yeast colonies due to the hydrolysis of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) by β-galactosidase was significantly delayed and less intense than the reaction we observed for strongly interacting mutants. The liquid β-galactosidase assay confirmed that the strength of the LcrG S23R-LcrV interaction was reduced (Table 2), indicating that this residue is likely involved in the interaction of LcrG with LcrV. As coiled-coil motifs show a characteristic seven amino acid repeat pattern, with each repeat representing two turns of the α-helix, it is interesting that three positions identified as important to the LcrG-LcrV interaction (16, 23, and 30) are each separated by seven amino acids. When this region was analyzed using a helical wheel projection, the three residues fall on the same face of the helix, which appears to be largely hydrophobic in nature (Fig. 3). These results and observations further indicate that the LcrG-LcrV interaction may require the participation of a coiled-coil motif and occurs at a largely hydrophobic interface.

To evaluate the biological significance of the changes in interaction seen in the yeast two-hybrid system between LcrV and LcrG S23R or LcrG L30R, LcrG S23R and L30R were expressed in lcrG Y. pestis where their ability to control Yops secretion was examined. Neither the S23R or the L30R mutant displayed the calcium-independent growth phenotype previously observed for LcrG A16R [20]. LcrG S23R displayed a wildtype growth and secretion phenotype while LcrG L30R was Ca²⁺-blind (data not shown). To further understand the phenotypes obtained, the ability of LcrG S23R and L30R to interact with LcrV was examined in Y. pestis. Wild type LcrG, LcrG S23R, LcrG L30R, and LcrG A16R were independently co-expressed with His₆-tagged LcrV in a strain of Y. pestis deleted for lcrGV. The cells were French pressed and the cleared lysates were applied to Talon columns to purify His₆-tagged LcrV. As previously reported [20], LcrG copurified with LcrV and LcrG A16R did not copurify with LcrV (Fig. 4, lanes 3 and 12). When LcrG S23R was coexpressed with His₆-tagged LcrV, a small amount was recovered in the elution fraction from the column (Fig. 4, lane 6). This result is indicative of a weaker interaction between LcrG S23R and LcrV than the interaction between wildtype LcrG and LcrV. The weaker interaction seen in Y. pestis between LcrG S23R and LcrV corresponds with our yeast two-hybrid data for the S23R mutant, which showed an interaction with LcrV, and suggests an interaction with decreased affinity (Table 2). The interaction results for LcrG S23R provide an explanation for the failure of LcrG S23R to confer a Ca²⁺-independent phenotype like the previously described LcrG A16R mutant. LcrG S23R still interacts with LcrV and therefore LcrV can neutralize the LcrG-mediated secretion block. In fact, this result is consistent with the fact that very low levels of LcrG are required to provide LcrG function [20]. LcrG L30R either interacts weakly with or does not interact with His₆-tagged LcrV in Y. pestis (Fig. 4, lane 9). This result also corresponds with the lack of interaction observed in the yeast two-hybrid assay for this mutant. Taken together, these results suggest that the residues found to be involved in the LcrG-LcrV interaction using two-hybrid analysis are relevant in the bacterial cell. However, the inability of L30R to function like our previously described A16R mutant was somewhat surprising. One explanation is that the protein with the L30R change is simply misfolded. The second explanation is that the secretion-blocking and the LcrV-interaction domains of LcrG overlap. Currently we do not possess the data required to differentiate between the two possibilities and we are pursuing the reasons for the failure of L30R to function like wildtype LcrG.

PcrG does not complement an lcrG strain, but interacts with LcrV

Because the residues of LcrG required for interaction with LcrV are strongly conserved in PcrG of P. aeruginosa (red asterisks in Fig. 2), we determined if PcrG could interact directly with LcrV. First, PcrG-GAL4-AD and LcrV-GAL4-BD chimeras were coexpressed in a yeast two-hybrid assay. Activation of β-galactosidase by the chimeric proteins was demonstrated by a filter-lift assay showing that LcrV and PcrG interact (data not shown). To determine if PcrG and LcrV also interacted in Y. pestis, we attempted to copurify the two proteins from bacterial extracts. PcrG and His₆-LcrV were coexpressed in a Y. pestis strain with lcrG and lcrV deleted and the cleared lysates were applied to a Talon column. PcrG reacts weakly with antibody to LcrG, allowing us to detect the presence of PcrG on immunoblots. PcrG was present in the elution fraction (Fig. 5A, lane 6), indicating that PcrG was retained on the Talon column. This result demonstrated that PcrG and LcrV interact in Y. pestis. The interaction of PcrG with LcrV supports the mutagenesis and sequence data indicating that residues conserved between LcrG and PcrG are likely to be important for interaction with LcrV.

Since PcrG and LcrV interacted in Y. pestis, the ability of PcrG to complement an lcrG strain of Y. pestis was examined. Cultures of lcrG strains transcomplemented with either LcrG or PcrG were grown in the presence and absence of calcium and harvested after four hours of growth at 37°C. Culture supernatants were examined for the presence of secreted proteins by immunoblotting for LcrV, YopE, and YopM. Fig. 5B shows that LcrG expression blocked secretion in this strain in the presence of calcium and allowed secretion in its absence (Fig. 5B, lanes 3 and 4). However, when PcrG was expressed, secretion was not blocked in either the presence or absence of calcium (calcium-blind phenotype) similar to the parent strain (Fig. 5B, lanes 5 and 6). Therefore, while PcrG is capable of interacting with LcrV, it differs from LcrG in its ability to block secretion of Yops in Y. pestis.

Selection of PcrG mutants that block secretion

PcrG of P. aeruginosa can interact with LcrV, but cannot block secretion of Yops in Y. pestis (Fig. 5B). This result suggested that mutagenesis of pcrG could be used to isolate PcrG mutants capable of blocking Yops secretion in Y. pestis. This genetic screen allowed the isolation of PcrG mutants that functioned in Y. pestis in a manner indistinguishable from LcrG (Fig. 5B, compare lanes 7 and 8 to lanes 3 and 4). DNA sequencing of seven independent isolates revealed the presence of a phenylalanine to leucine change at position 42 of PcrG in all seven of the isolates. This result suggests that a region of PcrG including amino acid 42 is capable of interaction with Y. pestis secretion components and suggests that the homologous LcrG region is likely to be involved in LcrG's secretion-blocking activity.

Analysis of the putative secretion-blocking region of LcrG

Change of residue 42 in PcrG from phenylalanine to leucine made the PcrG sequence look more like that of LcrG. That is, position 42 of PcrG became identical to the corresponding LcrG residue (L39) (Fig. 2, boxed residues). This information suggested that residue 39 of LcrG was an ideal candidate for site-directed mutagenesis to disrupt LcrG's secretion blocking activity. Accordingly, the leucine at position 39 in LcrG was mutated to both phenylalanine and arginine. Surprisingly, neither mutant LcrG protein L39F (data not shown) or L39R (Fig. 6B, lanes 5 and 6) effected LcrG function. Subsequent mutagenesis of the phenylalanine at position 48 of LcrG to arginine resulted in an LcrG mutant (F48R, blue asterisk in Fig. 2) that was defective for secretion blocking activity (Fig. 6B, lanes 7 and 8). Mutation of F48 to either alanine or leucine had no effect on the function of those LcrG variants, suggesting that the hydrophobic nature of F48 is important for LcrG function. The expression level of LcrG and LcrG F48R from our plasmid constructs were also analyzed in whole cell extracts. LcrG F48R was expressed as well as wild type LcrG, demonstrating that its inability to block secretion is not due to a change in the expression or stability of the protein (Fig. 6A, lanes 3–4 and 7–8). An interesting point to note is that when Skrzypek and Straley deleted residues 39–53 of LcrG (highlighted in blue in Fig. 2), they obtained a mutant protein that did not block Yops secretion [5]. The LcrG F48R mutant, like the Δ39–53 mutant, interacts with LcrV (data not shown and [4]), demonstrating that the protein likely folds in a native conformation. Additionally, the mutant confirms previous work that the two known LcrG functions (secretion blocking and LcrV interaction) are genetically separable [5]. Further experiments will more precisely delineate the region of LcrG required to block the type III secretion apparatus.

Bacterial strains, yeast strains, and growth conditions

DNA methods and plasmid construction

Plasmid DNA was isolated using a QiaPrep Spin kit (Qiagen, Inc., Studio City, CA). Cloning methods were essentially as described previously [31]. PCR fragments were purified using the QiaQuick PCR purification kit (Qiagen). Transformation of DNA into E. coli was accomplished by using commercially obtained competent cells (Novagen, Madison, WI). Electroporation of DNA into Y. pestis was done as described previously [32]. Gene amplification was performed with DeepVent (New England Biolabs, Beverly, MA) or Taq (Eppendorf Scientific, Westbury, NY) DNA polymerase in a Perkin-Elmer GeneAmp Model 2400 thermocycler (Applied Biosystems, Foster City, CA).

Site-directed mutagenesis was performed with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using the QuikChange Site-directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. Complementary oligonucleotides were designed to contain the desired mutation, flanked by unmodified sequence to anneal to the same sequence on opposite strands of the template plasmid. Oligonucleotide primers were synthesized by MWG Biotech (High Point, NC). pACTG was used as the template for all plasmids listed in Table 1 and Table 2. pAraG18K was used as the template for pJM99, pJM131, pJM143, and pJM144. All mutations were confirmed by sequence analysis (MWG Biotech). The sequences of all mutagenic oligonucleotides are available upon request.

Plasmids used in this study are described in Table 3. All plasmids expressing truncations of LcrG (pJM1, pJM2, pJM3, pJM4, pJM5, pJM23, pJM52, and pJM53) were constructed by cloning Bam HI- and Nco I-cleaved PCR products into pACT2. pJM110 was constructed by cloning a Bam HI- and Nco I-cleaved PCR product of pcrG into pACT2. Plasmid pJM127 was constructed by cloning an Eco RI-cleaved PCR p6roduct of pcrG into Eco RI- and Sma I-cleaved pBAD18. pJM127 was digested with Eco RI and Xba I to release a pcrG fragment that was subsequently subcloned into EcoRI- and XbaI-cleaved pBAD18-Kan to create pJM132. The sequences of all oligonucleotide primers are available upon request.

To facilitate the selection of PcrG variants that functioned to block Yops secretion (see below), the virulence plasmid, pCD1, of the lcrG Y. pestis strain KIM8-3002.7 was tagged with a kanamycin resistance cassette to select for its presence. pCD1 in KIM8-3002.7 was tagged with the mini-Tn5 derived transposon EZ::Tn™ <R6Kγori/KAN-2> (Epicentre Technologies, Madison WI) according to the manufacturer's instructions to obtain KIM8-3002.7.MN1. Location of the insertion was not determined but the LCR phenotype and ability to translocate Yops into HeLa cells was verified (data not shown).

Yeast two-hybrid assays

Yeast two-hybrid assays were performed as recommended by the commercial supplier (Clontech, Palo Alto, CA). Vectors pAS2-1 (encoding the GAL4 DNA binding domain) and pACT2 (encoding the GAL4 activation domain) were obtained from Clontech Laboratories as part of the Matchmaker Two-Hybrid System 2 Kit. Plasmids were transformed into Saccharomyces cerevisiae strain Y187 by using the Frozen-EZ Yeast Transformation II kit (Zymo research, Orange, CA) and plated on appropriate minimal yeast synthetic-dropout medium plates. Colony filter-lift assays to detect β-galactosidase activity were performed as described by Clontech laboratories. Liquid β-galactosidase assays were performed using the Yeast β-galactosidase Assay Kit (Pierce, Rockford, IL) according to the manufacturer's instructions. β-galactosidase units were calculated as described previously [30].

PcrG selection

Error-prone PCR was used to mutagenize pcrG for selection of PcrG variants that could block Yops secretion in an lcrG background (KIM8-3002.7.MN1). Mutazyme (Stratagene, La Jolla, CA) was used to amplify PcrG for cloning into pBAD18 [33]. The ligation reaction was electroporated into lcrG Y. pestis (which is temperature sensitive due to its Ca²⁺-blind phenotype) and the cells were plated on TMH medium [29] containing arabinose, Ca²⁺, kanamycin and carbenicillin. Transformants that grew at 37°C were streak purified and checked for phenotype by determining Ca²⁺-dependence, which suggests restoration of "LcrG function" by PcrG. Ca²⁺-dependent strains were subsequently used for plasmid isolation. Isolated plasmids were re-electroporated into an unmutagenized background and the strains were re-evaluated for growth at 37°C and secretion of Yops.

Cell fractionation and affinity purification

Bacterial cells were fractionated as previously described [18]. Briefly, bacterial cells were chilled on ice after growth, harvested by centrifugation, and washed in cold phosphate-buffered saline (PBS; [34]). Bacterial whole cell fractions were prepared by resuspending the washed cells in cold PBS and precipitating total proteins with 10% (vol/vol) trichloroacetic acid (TCA) on ice overnight. Secreted proteins were recovered from the bacterial growth medium by centrifuging the spent medium a second time, transferring the supernatant to a clean tube, and precipitating with 10% (vol/vol) TCA on ice overnight. The TCA-precipitated proteins were pelleted by centrifugation (20,800 × g at 4°C) for 20 min and resuspended in 2X sodium dodecyl sulfate (SDS) sample buffer [34]. Protein extracts for affinity-purification were prepared by disintegration with a French press. Ice-cold yersiniae were resuspended in ice-cold PBS and passed through a French pressure cell at 20,000 psi. Following disintegration, the extracts were clarified by centrifugation at 20,800 × g for 10 min at 4°C. Affinity purification with His₆-LcrV was performed using Talon resin (Clontech) as described by the manufacturer.

Protein electrophoresis and immunodetection

Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5 % (wt/vol) polyacrylamide gels according to the method of Laemmli [35]. Samples were boiled 3–5 min before loading on the gels. Samples were loaded such that lanes containing different culture fractions represented equivalent amounts of the original cultures. Proteins resolved by SDS-PAGE were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) using carbonate transfer buffer (pH 9.9) [5]. Specific proteins were visualized using rabbit polyclonal antibodies specific for glutathione S-transferase (GST)-tagged LcrG (α-LcrG) [4], His-tagged LcrV (α-LcrV) [4], YopM (α-YopM) [36], GST-tagged LcrQ (α-LcrQ) [16], YopN (aka LcrE) (α-LcrE [4]), His-tagged YopD (α-YopD) [16], and YopE (α-YopE; gift from S. C. Straley, University of Kentucky, Lexington, KY. Alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G; Pierce) was used to visualize proteins by development with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Fisher Scientific, Fair Lawn, NJ).