Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface
© Matson and Nilles; licensee BioMed Central Ltd. 2002
Received: 1 May 2002
Accepted: 28 June 2002
Published: 28 June 2002
Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.
In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.
Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.
Yersinia pestis, the causative agent of bubonic plague, harbors a large virulence plasmid that is essential for pathogenicity. This plasmid, called pCD1  in Y. pestis, encodes for a type III secretion apparatus necessary for the translocation of effector proteins (Yops) into eukaryotic target cells . The net result of this polarized toxin translocation is the disruption of eukaryotic cell signalling and structure . Induction of the virulence-related genes on pCD1 is linked to an in vitro phenomenon termed the Low Calcium Response (LCR). The LCR is believed to mimic the response of Y. pestis in contact with eukaryotic cells when calcium ions are removed from the growth medium at 37°C. The result of this in vitro induction of virulence genes is the massive secretion of Yops into the growth medium, accompanied by a characteristic growth restriction that is a hallmark phenotype of the LCR .
Secretion of Yops by the type III secretion system is not constitutive. Control of secretion is complex and involves several proteins that positively and negatively regulate the activity of the secretion apparatus in response to environmental conditions . LcrG has been implicated in blocking the secretion apparatus either directly or indirectly , as strains lacking lcrG constitutively secrete Yops , regardless of inducing conditions. In addition, YopN, TyeA, SycN, YscB, LcrH, YopD, and LcrQ are all also required for the negative regulation of Yops secretion [6–17]. LcrV (the V antigen) is thought to positively regulate Yops secretion as lcrV strains do not secrete Yops, even under conditions that normally favor secretion [18, 19].
LcrG and LcrV have been demonstrated to physically interact in the bacterial cytoplasm . Disruption of the LcrG-LcrV interaction or overexpression of LcrG in a low-LcrV background both result in a constitutive blockage of Yops secretion [18, 20]. This evidence led to a model of how these two proteins may control Yops secretion. The LcrG-titration model theorizes that LcrG blocks the secretion apparatus (Ysc) from the bacterial cytoplasm and that interaction with LcrV is required to relieve the blockage. Upon induction by eukaryotic cell contact or removal of calcium from the growth medium, the intracellular level of LcrV becomes elevated relative to that of LcrG. The increased amount of LcrV removes LcrG from its secretion-blocking role, resulting in full induction of the LCR and secretion of Yops. Evidence accumulated to date clearly demonstrates that the LcrG-LcrV interaction is critical for the regulation of this complex virulence system [18, 20]. Therefore, dissection of the LcrG-LcrV interaction is warranted.
Utilizing yeast two-hybrid analyses, we further investigated the LcrG-LcrV interaction. We determined that the N-terminus of LcrG is essential for LcrV interaction. The smallest linear region of LcrG required for the interaction is predicted to form a coiled-coil motif, a structure often associated with protein-protein interactions. We used both alanine-scanning and a 'radical' site-directed mutagenesis strategy to identify amino acids critical for the interaction within this region of LcrG. While alanine mutants failed to disrupt the LcrG-LcrV interaction, radical mutations of three residues revealed a region important for the interaction. These residues are likely to reside on the same face of the protein if a coiled-coil structure is assumed. Based on this evidence, we predict that the interaction of LcrG with LcrV occurs within an N-terminal coiled-coil domain of LcrG via hydrophobic interactions.
Additionally, we investigated whether the Pseudomonas homolog of LcrG, PcrG, could transcomplement an lcrG strain of Y. pestis. The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system for intracellular delivery of toxins [21–23] that is the most similar to the Yersinia system . Several components of the Pseudomonas system have been demonstrated to functionally complement genetic defects in the Yersinia homologs including the LcrV homolog, PcrV [24, 25]. Homologs of LcrG and LcrV (i.e., PcrG and PcrV) have only been identified in P. aeruginosa, suggesting that this pair of regulatory proteins may fulfill similar roles in these extracellular pathogens. In the present study, we found that PcrG could not substitute for LcrG, although both proteins interacted with LcrV. This result led to a genetic selection for PcrG variants that could transcomplement an lcrG defect. Isolation of a PcrG variant that could substitute for LcrG led to the identification of a region within LcrG that may be involved in blocking the activity of the Y. pestis type III secretion system.
Results and Discussion
Delineation of the LcrV-interaction domain of LcrG
Alanine-scanning mutagenesis of the LcrG N-terminus
Two hybrid analysis of alanine site-directed mutants of LcrG (residues 7–40) with LcrV.
68.05 ± 2.96
6.71 ± 0.51
51.55 ± 5.15
50.80 ± 5.13
58.60 ± 2.57
54.85 ± 6.19
59.55 ± 0.95
51.85 ± 3.75
42.65 ± 0.85
66.73 ± 1.80
45.93 ± 3.91
43.27 ± 2.67
47.87 ± 3.43
47.00 ± 2.99
47.90 ± 3.30
65.25 ± 4.85
48.40 ± 3.00
46.73 ± 2.00
52.27 ± 2.23
'Radical' site-directed mutagenesis of LcrG allows the LcrG-LcrV interaction to be disrupted
Two hybrid analysis of 'radical' site-directed mutants of LcrG (residues 7–40) with LcrV.
68.05 ± 2.96
6.71 ± 0.51
83.09 ± 5.12
55.10 ± 4.31
4.45 ± 0.42
4.07 ± 0.22
66.03 ± 4.94
86.61 ± 8.92
36.44 ± 3.72
14.30 ± 1.61
21.99 ± 1.24
53.72 ± 3.54
1.91 ± 0.26
52.42 ± 2.10
35.66 ± 2.38
PcrG does not complement an lcrG strain, but interacts 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
The interaction of LcrG with LcrV is a critical factor in controlling the activity of the type III secretion system in Y. pestis. In order to gain a deeper understanding of this critical interaction, we determined the region of LcrG required for interaction with LcrV. Results presented in this study demonstrate that the smallest linear region of LcrG required for interaction with LcrV is residues 7 to 40 (highlighted in yellow in Fig. 2). Interestingly, this region is predicted to form a coiled-coil domain. We have identified several residues (indicated with red asterisks in Fig. 2) within the interacting region of LcrG that contribute to the interaction with LcrV. When residues 7 to 40 of the protein are analyzed with a helical wheel projection, these three residues (A16, S23, and L30) appear to lie on one face of an amphipathic helix (Fig. 3). The seven amino-acid periodicity of the residues is consistent with a coiled-coil protein motif. These data strongly suggest the LcrV-interaction domain of LcrG is contained within an amphipathic coiled-coil domain. Additionally, we exploited the inability of PcrG to block secretion in Y. pestis as a point to start mapping the region of LcrG required for blocking the type III secretion apparatus. We found that mutation of residue 42 in PcrG (boxed in Fig. 2) allowed the protein to function in Y. pestis. This information, coupled with a previous observation that deletion of residues 39 to 53 within LcrG (highlighted in blue in Fig. 2) abolished the secretion-blocking activity , allowed us to identify amino acid 48 as a residue (blue asterisk in Fig. 2) required to block the activity of the Y. pestis type III secretion apparatus.
Materials and Methods
Bacterial strains, yeast strains, and growth conditions
Strains and plasmids used in this studya.
Strain or plasmid
Source or reference
recA1 endA1 hsdR17 (rK- mK+) supE44 thi-1 gyrA96 relA1 lac [F' proA+B+lacIqZ ΔM15::Tn10]
MATa ura3-52 his3-200 ade2-101 trp1-901 leu2-3 112 gal4 Δmet gal80 ΔURA3::GAL1UAS-GAL1TATA-lacZ
GAL4(768–881) AD LEU2 Apr
pACT2 + lcrG
GAL4(1–147) DNA-BD TRP1 AprCYHS2
pAS2-1 + lcrV
pACT2 + lcrG' 1–40b
pACT2 + lcrG' 34–70b
pACT2 + lcrG' 64–95b
pACT2 + lcrG' 1–70b
pACT2 + lcrG' 34–95b
pACT2 + lcrG' 1–33b
pACT2 + lcrG' 7–40b
pACT2 + lcrG' 12–40b
araBADp cloning vector, Kmr
pBAD18-Kan + lcrG
pBAD24 + His6-LcrV, Apr
pBAD18-Kan + lcrG (A16R)c
pBAD18-Kan + lcrG (L30R)c
pBAD18-Kan + lcrG (S23R)c
pACT2 + pcrG
pBAD18 + pcrG
pBAD18-Kan + pcrG
pBAD18 + pcrG (F42L)c
pBAD18-Kan + lcrG (L39R)c
PBAD18-Kan + lcrG (F48R)c
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 . 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 . 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 .
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 . 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  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 . Briefly, bacterial cells were chilled on ice after growth, harvested by centrifugation, and washed in cold phosphate-buffered saline (PBS; ). 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 . 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 . 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) . Specific proteins were visualized using rabbit polyclonal antibodies specific for glutathione S-transferase (GST)-tagged LcrG (α-LcrG) , His-tagged LcrV (α-LcrV) , YopM (α-YopM) , GST-tagged LcrQ (α-LcrQ) , YopN (aka LcrE) (α-LcrE ), His-tagged YopD (α-YopD) , 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).
This work was supported in part by funds from North Dakota EPSCoR and the UND School of Medicine. J. S. M. was supported by a pre-doctoral fellowship from ND EPSCoR. The authors thank David S. Bradley (University of North Dakota) for critical reading of the manuscript.
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