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J. Biol. Chem., Vol. 275, Issue 47, 36869-36875, November 24, 2000

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LcrV, a Substrate for Yersinia enterocolitica Type III Secretion, Is Required for Toxin Targeting into the Cytosol of HeLa Cells^*

Vincent T. Lee, Christina Tam, and Olaf Schneewind^§

From the Department of Microbiology and Immunology, University of California Los Angeles School of Medicine, Los Angeles, California 90095

Received for publication, March 22, 2000, and in revised form, July 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pathogenic Yersinia species employ type III machines to transport virulence factors across the bacterial envelope. Some substrates for the type III machinery are secreted into the extracellular medium, whereas others are targeted into the cytosol of host cells. We found that during infection of tissue culture cells, yersiniae secrete small amounts of LcrV into the extracellular medium. Knockout mutations of lcrV abolish Yersinia targeting and reduce expression of the lcrGVHyopBD operon. In contrast, a block in LcrV secretion does not affect targeting, but results in premature expression and secretion of Yop proteins into the extracellular medium. LcrV-mediated activation of the type III pathway is thought to occur by sequestration of the regulatory factor LcrG, presumably via the formation of LcrV·LcrG complexes. These results suggest that intrabacterial LcrV regulates the expression and targeting of Yop proteins during Yersinia infection, whereas secreted LcrV is required to ensure specificity of Yop injection into eukaryotic cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pathogenic Yersinia species colonize the lymphoid tissues of their infected hosts and require the type III machinery to escape phagocytic killing by immune cells (1, 2). Bacterial entry into the host triggers the synthesis and assembly of the type III machinery (3-5). When exposed to high calcium concentrations within extracellular fluids, the type III pathway of yersiniae is thought to be inactive (6, 7). Bacterial attachment to host cells activates the type III machinery, causing the secretion of YopBDR (Yersinia outer protein) into the extracellular medium (8-10) and the targeting of YopEHMNOPT (effector Yop proteins) into the eukaryotic cytosol (8, 11-16). The synthesis of effector Yop proteins is regulated, and bacterial attachment to host cells results in a large increase in yop gene expression (17). Once within the eukaryotic cytosol, effector Yop proteins interfere with macrophage signal transduction and actin polymerization (18-20), thereby preventing bacterial phagocytosis and inducing the apoptotic demise of immune cells (11, 16, 21).

The genes specifying type III machinery components, Yop proteins, as well as regulatory factors are located on a 70-kilobase virulence plasmid (2). Loss of the virulence plasmid or of type III machinery genes causes dramatic defects in the pathogenesis of Yersinia infections (22). Bacterial growth at 37 °C and the chelation of calcium from the culture medium artificially activate the Yersinia type III pathway, leading to the massive secretion of 14 proteins (YopBDEHMNOPQRT, LcrV (low calcium response), and YscM1/YscM2 (LcrQ)) into the surrounding medium (yop) (23-25). Loss-of-function mutations in any one of the 22 ysc (Yop secretion) genes abrogate all protein export by the type III pathway (26-29). Knockout mutations of lcrV do not interfere with the secretion of Yop proteins under low calcium conditions (30), but abolish the targeting of effector Yop proteins into the cytosol of eukaryotic cells (31). In contrast, lcrG mutant yersiniae secrete Yop proteins even in the presence of calcium, suggesting that LcrG may regulate the expression or secretion of Yop proteins (32-34). Straley and co-workers (35) observed LcrG binding to LcrV in the bacterial cytoplasm and proposed a model whereby LcrG may physically block the type III machinery. Binding to LcrV is thought to interfere with the regulatory activity of LcrG, thereby activating the type III pathway (titration model) (35). Recent observations suggest that LcrG functions as a chaperone, promoting type III secretion of LcrV under conditions in which the secretion of effector Yop proteins is blocked (5 mM calcium).¹

Using immunofluorescence microscopy, Wolf-Watz and co-workers (37) observed LcrV staining on the surface of Yersinia pseudotuberculosis. The addition of LcrV antiserum to tissue culture cells prevents the type III injection of effector Yop proteins (37). Straley and co-workers (31, 38) examined the fate of LcrV during Yersinia pestis infection and reported that LcrV may be surface-exposed as well as injected into the cytosol of HeLa cells. These investigators observed no inhibition of type III targeting following the addition of LcrV antiserum to infected tissue culture cells (38). lcrV mutant yersiniae have been reported to be defective in the type III secretion of YopB and YopD (30). Cornelis (39) proposed a mechanism whereby the formation of an LcrV·YopB·YopD complex allows type III export and assembly of an injectisome on the bacterial surface. The Yersinia injectisome, presumably composed of many proteins in addition to LcrV·YopB·YopD, is thought to insert into the plasma membrane of eukaryotic cells, thereby catalyzing the translocation of effector Yop proteins (39). Nevertheless, the Yersinia injectisome has not yet been purified or viewed by microscopy.

We have examined the subcellular location of LcrV during Yersinia enterocolitica infection of HeLa cell cultures using the digitonin fractionation technique (8). When incubated in tissue culture medium with or without HeLa cells, yersiniae secrete YopBDR and LcrV into the extracellular medium even in the presence of 1.8 mM calcium. During infection of HeLa cultures, lcrV mutant yersiniae secrete YopBDR into the extracellular medium, but fail to inject YopEHMN. This defect is complemented by plasmid-encoded wild-type lcrV and, at least in part, by GST²-lcrV, specifying a hybrid protein that cannot be exported by the type III pathway. GST-LcrV copurifies with LcrG, but not with YopB or YopD. Expression of GST-LcrV in lcrV mutant yersiniae causes the same Los (loss of type III targeting specificity) phenotype as knockout mutations of lcrG, resulting in premature activation of the type III targeting pathway and in secretion of all Yop proteins into the extracellular medium. Thus, intrabacterial LcrV is essential for the type III targeting pathway, whereas secreted LcrV appears to ensure the fidelity of the Yop protein injection process.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Plasmids-- Y. enterocolitica strain W22703 (wild type) has been previously described (40). Y. enterocolitica strain CT1 (lcrV) was constructed by allelic exchange using the suicide plasmid pLC28 (41). The lcrV1 allele was designed as a mutation that introduced a stop codon at position 11 of the lcrV open reading frame (42, 43), followed by a +1 reading frameshift and a unique EcoRI site as a fusion joint between two DNA fragments. lcrV1 was constructed from two PCR products that were amplified using primers LcrV.1 (5'-AAGAGCTCACCAGTGCAGGGAGTTATTT-3') and LcrV.2 (5'-AAGAATTCACTATTGTGGGTTTTGTTCGTAGGC-3') as well as primers LcrV.3 (5'-AAGAATTCACATTTATTGAGGATCTAGAAAA-3') and LcrV.4 (5'-AACTGCAGTGAGTGTCTGTCGTCTCTTG-3'). The PCR products were cut with SacI-EcoRI or EcoRI-PstI, fused at the EcoRI site, and cloned between the SacI-PstI sites of pLC28. To generate pVL49, the lcrV open reading frame was PCR-amplified with two primers carrying abutted NdeI and BamHI restriction sites: LcrV.5 (5'-AACATATGATTAGAGCCTACGAACAA-3') and LcrV.6 (5'-AAGGATCCTCATTTACCAGACGTGTCATC-3'). The PCR product was digested with NdeI-BamHI and cloned between the NdeI and BamHI sites of the low-copy-number plasmid pVL41 (9) to generate pVL49. pVL47 was generated by PCR amplification using primers LcrV.6 and LcrV.7 (5'-AAGGTACCATTAGAGCCTACGAACAAAACC-3'). lcrV sequences were cut with KpnI-BamHI and cloned into pDA255 (44). Expression of LcrV and GST-LcrV is under the control of the tac promoter. The lacI^Q allele is also cloned on the low-copy-number vector, and Y. enterocolitica transformants were induced for expression of LcrV and GST-LcrV by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside. To raise LcrV-specific antibody, the N-terminal domain of LcrV was appended to an affinity tag and purified. lcrV coding sequence was PCR-amplified with primers that carry abutted BamHI sites (LcrV.8, 5'-AAGGATCCATGGCTTACGACCTTTCTGAG-3'; and LcrV.9, 5'-AAGGATCCAATTTTTAATTCGGCGGTAAGC-3'). The PCR product was digested with BamHI and cloned into pQE30 (QIAGEN Inc.), thereby generating pVL1067.

Yersinia Secretion-- Yersiniae were grown in tryptic soy broth supplemented with 5 mM calcium chloride or 5 mM EGTA. Overnight cultures of Yersinia were diluted 1:50 into 30 ml of fresh medium, grown for 2 h at 26 °C, and induced at 37 °C for 3 h. Cultures were centrifuged at 10,500 × g for 15 min, and the supernatant was separated from the cell pellet. Proteins in both fractions were precipitated with trichloroacetic acid, washed with acetone, and suspended in sample buffer. Proteins were analyzed by SDS-PAGE and immunoblotting. Immunoreactive species were quantified as chemiluminescent signals on x-ray film using laser densitometry scanning.

Yersinia Infection of HeLa Cell Cultures-- Overnight cultures of Yersinia were diluted 1:20 into 30 ml of fresh Luria broth and grown for 2 h at 26 °C with shaking. Bacteria were sedimented at 8000 × g for 10 min and suspended in phosphate-buffered saline. HeLa cells were grown to 80% confluency in 75-cm² tissue culture flasks with Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum. Prior to infection, cells were washed twice with PBS, covered with 10 ml of DMEM, and warmed to 37 °C for 30 min. Aliquots of HeLa cells were counted, and each flask was infected with yersiniae at a multiplicity of infection of 10 and incubated for 3 h at 37 °C with 5% CO₂. The culture medium was removed and centrifuged at 32,000 × g for 15 min to separate soluble proteins from non-adherent bacteria in the sediment. HeLa cells as well as adherent bacteria were scraped off the flasks into 10 ml of digitonin in PBS and placed on a rotary shaker for 20 min. Samples were centrifuged at 32,500 × g for 15 min. A 7-ml aliquot was withdrawn and precipitated with chloroform/methanol, whereas the remaining supernatant was discarded. The sediment was suspended in 10 ml of PBS, and a 7-ml aliquot was precipitated with chloroform/methanol. Protein precipitates were solubilized in sample buffer, separated on SDS-polyacrylamide gel, and analyzed by immunoblotting with specific antiserum. Immunoreactive species were quantified as chemiluminescent signals on x-ray film using laser densitometry scanning.

Purification of LcrV-- Escherichia coli XL1-Blue/pVL1067 cells (1 liter) were grown to mid-log phase at 37 °C and induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 3 h. Cells were harvested by centrifugation at 8000 × g for 15 min and suspended in 20 ml of buffer A (6 M guanidine hydrochloride, 0.1 M NaH₂PO₄, and 0.01 M Tris-HCl, pH 8.0). The sample was incubated with intermittent vortexing on ice for 1 h. Insoluble material was removed by centrifugation at 32,000 × g for 10 min. The supernatant was loaded onto a 1-ml column of nickel-nitrilotriacetic acid-Sepharose that had been pre-equilibrated with 10 ml of buffer A. The column was washed with 10 ml of buffer A, 10 ml of buffer B (8 M urea, 0.1 M NaH₂PO₄, and 0.01 M Tris-HCl, pH 8.0), and 20 ml of buffer C (same as buffer B, but pH 6.3). LcrV-His₆ was eluted with 4 ml of buffer D (same as buffer B, but pH 4.5). Samples were aliquoted and stored frozen at 80 °C. Purified LcrV was emulsified with Freund's adjuvant and injected into rabbits to raise polyclonal antibodies. Antiserum reactivity and specificity were examined by comparing bacterial extracts of wild-type and mutant lcrV strains with the purified antigen.

Affinity Chromatography-- Overnight cultures of Y. enterocolitica CT1/pVL47 were diluted 1:50 into fresh tryptic soy broth supplemented with 30 µg/ml chloramphenicol. Bacteria were grown and induced by incubation for 2 h at 26 °C and for 3 h at 37 °C. Cells from 500 ml of culture were harvested by centrifugation at 8000 × g for 15 min. The cell pellet was suspended in 20 ml of buffer E (50 mM Tris-HCl, 20% sucrose, and 1 mM dithiothreitol, pH 7.0), and bacteria were broken by two passages through a French pressure cell at 14,000 p.s.i. Unbroken cells and debris were removed by centrifugation at 32,500 × g for 15 min. The supernatant, containing soluble cytoplasmic extract, was subjected to affinity chromatography on glutathione-Sepharose. The column was washed with 30 column volumes of column buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.5), and proteins were eluted with 4 ml of column buffer containing 10 mM glutathione. Eluted proteins were mixed with an equal volume of sample buffer containing 3 M urea and analyzed by SDS-PAGE and immunoblotting.

Microscopy-- HeLa cells were grown in DMEM and 10% fetal bovine serum on glass coverslips in a 24-well plate for 48 h at 37 °C. Coverslips were washed with PBS and incubated with DMEM prior to infection with Y. enterocolitica strains W22703/pDA37 (wild type, expressing plasmid-encoded Npt (neomycin phosphotransferase)), CT1/pDA37 (lcrV, expressing plasmid-encoded Npt), CT1/pVL49 (expressing plasmid-encoded wild-type LcrV), and CT1/pVL47 (expressing plasmid-encoded GST-LcrV) for 3 h at an multiplicity of infection of 20. After infection, coverslips were fixed with 3.7% formaldehyde for 10 min. All fixation was quenched by adding 0.1 M glycine for 10 min, followed by permeabilization with 0.1% Triton X-100 for 10 min. Samples were blocked with 3% bovine serum albumin in PBS for 30 min. Coverslips were then incubated with anti-YopE polyclonal antibodies for 45 min. After washings with PBS, samples were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc.) for 45 min. Samples were washed and viewed under a fluorescence microscope. Images were captured with a Hamamatsu CCD camera.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Secretion of LcrV into the Extracellular Medium-- To determine the fate of LcrV during infection, HeLa cell cultures were infected with Y. enterocolitica W22703. The medium was decanted and centrifuged, separating non-adherent bacteria from the extracellular medium. HeLa cells and adherent yersiniae were extracted with digitonin, thereby disrupting the cholesterol-containing plasma membrane of eukaryotic cells. The bacteria were sedimented by centrifugation and separated from the cytosol of HeLa cells. Proteins in all fractions were precipitated with chloroform/methanol and analyzed by immunoblotting (8). Digitonin extraction released p130^Cas (45) from the cytosol of HeLa cells. Npt, a protein residing in the Yersinia cytoplasm (46), was not released and sedimented with the bacteria. The Yersinia type III machinery secreted YopBDR into the extracellular medium and targeted YopEH into the cytosol of HeLa cells. Small amounts of LcrV were found in the extracellular medium; however, the majority of LcrV sedimented with yersiniae after digitonin extraction of HeLa cells (Fig. 1A and Table I). LcrV was not detected in the supernatant of digitonin-extracted HeLa cells, suggesting that LcrV is not injected into the cytosol of eukaryotic cells.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Yersiniae secrete YopBDR and LcrV into the extracellular medium. A, infection of HeLa cells with Y. enterocolitica W22703. HeLa cells were infected for 3 h. The tissue culture medium (Med) was decanted and centrifuged to separate secreted proteins (supernatant (S)) from those present within non-adherent bacteria (pellet (P)). HeLa cells and adherent yersiniae were extracted with digitonin (Dig), a detergent that solubilizes the eukaryotic plasma membrane, but not the bacterial envelope. Extracts were centrifuged to separate proteins solubilized from the HeLa cytoplasm (supernatant) from those that sedimented with the bacteria (pellet). Proteins in each fraction were precipitated with chloroform/methanol and analyzed by SDS-PAGE and immunoblotting. p130Cas is located in the cytoplasm of HeLa cells, whereas Npt is located in the cytoplasm of yersiniae. Y. enterocolitica W22703 injected YopE and YopH into HeLa cells, whereas YopBD and LcrV were secreted into the extracellular medium. B, Y. enterocolitica W22703 was incubated in tissue culture medium (DMEM with 0.2% fetal bovine serum and 1.8 mM calcium) and incubated for 3 h at 37 °C. Cultures were centrifuged to separate proteins secreted into the extracellular medium with the supernatant from those that sedimented into the bacterial pellet. Samples were precipitated with trichloroacetic acid and analyzed by SDS-PAGE and immunoblotting. In the presence of 1.8 mM calcium, yersiniae secreted YopBDR and LcrV into the extracellular medium, whereas YopEH and Npt were not secreted. See Table I for quantification of the data.

LcrV Secretion Occurs without Bacterial Attachment to HeLa Cells-- To examine whether YopBDR and LcrV are secreted prior to the attachment of yersiniae to eukaryotic cells, tissue culture medium (DMEM containing 0.2% fetal bovine serum and 1.8 mM calcium) was inoculated with Y. enterocolitica W22703. After incubation for 3 h, cultures were centrifuged, and the supernatant and pellet fractions were precipitated with trichloroacetic acid and analyzed by immunoblotting. YopBDR and LcrV were found in the tissue culture medium, indicating that these proteins had been secreted even without bacterial attachment to HeLa cells (Fig. 1B and Table I). YopE and YopH were not found in the tissue culture medium, but sedimented with the bacteria. This result is consistent with the notion that export of type III targeting substrates requires bacterial attachment to eukaryotic cells (11). As a control, Npt sedimented with the bacteria into the pellet fraction.

LcrV Is Required for the Calcium Regulation of the lcrGVHyopBD Operon-- A knockout mutation of lcrV was generated by introducing a stop codon and +1 frameshift mutation at position 11 of the lcrV open reading frame. To examine the role of LcrV in type III secretion, Y. enterocolitica strains W22703 (wild type) and CT1 (lcrV) were grown at 37 °C in the presence or absence of 5 mM calcium. Cultures were centrifuged, and the supernatant and pellet fractions were precipitated with trichloroacetic acid and analyzed by immunoblotting (Fig. 2). When induced by low calcium, wild-type yersiniae secreted YopBDE and LcrV into the extracellular medium. As a control, LcrG and LcrH, two bacterial chaperones, were found in the sediment of all cultures examined. The addition of calcium reduced the expression and type III secretion of YopBDE. In contrast, LcrV was found secreted equally in the presence and absence of calcium. The lcrV mutant strain CT1 expressed less LcrGH and YopBD than did wild-type yersiniae both in the presence and absence of calcium (Table II). Nevertheless, in the absence of calcium, YopBD were secreted at a level similar to that observed for the wild-type strain. Transformation of Y. enterocolitica CT1 with pVL49, encoding wild-type lcrV, not only restored LcrV expression and secretion, but also increased the expression of the lcrGVHyopBD operon (Table II). The data suggest that lcrV knockout mutations result in reduced expression of the lcrGVHyopBD operon without affecting YopBD secretion. These data are in disagreement with the previous report that LcrV may be required for the secretion of YopB and YopD (30).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   LcrV is required for the low calcium regulation of the lcrGVHyopBD operon. Y. enterocolitica strains W22703 (wild type (WT)) and CT1 (lcrV) were transformed with pDA37 (vector control), pVL49 (lcrV), or pVL47 (GST-lcrV) and grown in the presence or absence of calcium for 3 h at 37 °C. Cultures were centrifuged, and the supernatant (S) was separated from the cell pellet (P). Protein in each sample was precipitated with trichloroacetic acid, solubilized in sample buffer, and analyzed by SDS-PAGE and immunoblotting. See Table II for quantification of the data. CAT, chloramphenicol acetyltransferase.

Intrabacterial LcrV Induces the lcrGVHyopBD Operon-- Fusion of Yop proteins to the C terminus of glutathione S-transferase abolishes their transport by the type III machinery (9). We wondered whether the secretion of LcrV could also be blocked by fusion to GST. Plasmid pVL47, encoding GST-LcrV, was transformed into Y. enterocolitica CT1. When expression of GST-LcrV was induced by the addition of isopropyl-1-thio--D-galactopyranoside, Y. enterocolitica CT1 synthesized and secreted YopBDE at a level similar to wild-type yersiniae (Fig. 2). GST-LcrV was not secreted into the extracellular medium, indicating that fusion to glutathione S-transferase indeed blocked transport of LcrV by the type III machinery (Fig. 2). Expression of GST-LcrV abolished the calcium repression of lcrGVHyopBD and caused Y. enterocolitica CT1/pVL47 to secrete large amounts of Yop proteins even in the presence of calcium (Table II). Thus, a block in the secretion of LcrV appears to induce the expression of lcrGVHyopBD and to activate the type III pathway.

Formation of Intracellular GST-LcrV·LcrG Complexes-- To purify GST-LcrV from yersiniae, crude extracts of Y. enterocolitica CT1/pVL47 were centrifuged to remove cellular debris. Soluble components of Yersinia cell extracts were subjected to affinity chromatography on glutathione-Sepharose. Samples were analyzed by Coomassie Blue-stained SDS-PAGE, revealing copurification of GST-LcrV and LcrG (Fig. 3A). After immunoblotting, immunoreactive signals in the eluate and load fraction were quantified, and the eluate/load ratio was determined. Eluate/load ratios of 1.54 for LcrG and 1.93 for LcrV indicated enrichment of both polypeptides during affinity chromatography. These data confirmed previous observations that LcrV and LcrG form a complex in the cytosol of yersiniae (35). Neither YopB nor YopD eluted with GST-LcrV, indicating that these two polypeptides did not bind to LcrV. This was a surprising result, as LcrV has been reported to interact with both YopB and YopD (30). Failure of GST-LcrV to interact with YopB and YopD is likely not caused by the fusion of GST, as purification of YopD from the cytoplasm of yersiniae also did not yield copurified LcrV.³

View larger version (76K): [in this window] [in a new window]   Fig. 3.   GST-LcrV binds to LcrG. A, Y. enterocolitica CT1 was transformed with pVL47 (GST-LcrV) and grown in tryptic soy broth at 37 °C. GST-LcrV was expressed by inducing the tac promoter with 1 mM isopropyl-1-thio--D-galactopyranoside. Cells were disrupted in a French pressure cell. Extracts were centrifuged to remove insoluble material, and the cleared lysate (L) was subjected to affinity chromatography on glutathione resin. Column flow-through (FT), wash (W), and eluate (E) were collected and analyzed by Coomassie Blue-stained SDS-PAGE. Arrowheads mark the migration of GST-LcrV (black) and LcrG (white). The positions of molecular mass markers (in kilodaltons) are indicated. B, the eluate and load (L) samples were analyzed by immunoblotting. To quantify binding of GST-LcrV to LcrG, we determined the relative concentration of protein in the eluate and load by scanning of immunoreactive signals. A eluate/load protein concentration ratio of >1 reveals enrichment of proteins during the purification. GST-LcrV copurified with LcrG, but not with LcrH, YopB, YopD, or chloramphenicol acetyltransferase (CAT).

LcrV Is Required for the Type III Targeting of Effector Yop Proteins-- To examine the role of LcrV in type III targeting, HeLa cells were infected with Y. enterocolitica strains W22703 (wild type) and CT1 (lcrV). Wild-type yersiniae targeted YopEHMN into the cytosol of HeLa cells (Fig. 4). YopBDR and small amounts of LcrV were secreted into the extracellular medium. Most of LcrV as well as YopQ remained with the bacterial sediment after digitonin extraction of HeLa cells (Fig. 4). The lcrV mutant strain expressed very little YopBDEHMN, LcrGH, and SycE (Fig. 5). Moreover, lcrV mutant yersiniae failed to inject YopEHMN into the cytosol of HeLa cells (Fig. 5). Instead, small amounts of YopEHMN were secreted into the extracellular medium. Transformation of Yersinia CT1 with plasmid pVL49 (encoding wild-type lcrV) increased the expression of the yop virulon and the targeting of YopEHMN to wild-type levels (Fig. 5).

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Overexpression of LcrV and GST-LcrV in wild-type yersiniae causes an up-regulation of the type III pathway. Y. enterocolitica W22703 was transformed with pDA37 (vector control), pVL49 (LcrV), or pVL47 (GST-LcrV). HeLa cells were infected with yersiniae and subjected to digitonin (Dig) fractionation and immunoblotting as described in the legend to Fig. 1. Yersiniae overexpressing LcrV or GST-LcrV displayed an up-regulation of the type III pathway as observed for the increased expression of YopBDEHMNR. Med, tissue culture medium; S, supernatant; P, pellet; FPT, farnesyl protein transferase, acytosolic control for the permeabilization of HeLa cells.

View larger version (57K): [in this window] [in a new window]   Fig. 5.   lcrV mutant yersiniae fail to inject YopEHMN into HeLa cells. Y. enterocolitica CT1 (lcrV) was transformed with pDA37 (vector control), pVL49 (LcrV), or pVL47 (GST-LcrV). HeLa cells were infected with yersiniae and subjected to digitonin (Dig) fractionation and immunoblotting as described in the legend to Fig. 1. The lcrV mutant strain CT1 failed to target YopEHMN into HeLa cells (Not (no type III targeting) phenotype). Moreover, the expression of Yop proteins was reduced compared with the wild-type parent strain (see Fig. 4). Expression of plasmid-encoded LcrV in the lcrV mutant strain CT1 restored type III targeting to wild-type levels. Furthermore, expression of the non-secretable GST-LcrV in the lcrV mutant strain CT1 not only restored type III targeting, but also caused massive secretion of YopEHMN into the extracellular medium (Los phenotype). Med, tissue culture medium; S, supernatant; P, pellet; FPT, farnesyl protein transfer.

Intrabacterial LcrV Complements the Targeting Defect of lcrV Mutant Yersiniae-- Previous work suggested that LcrV may perform an essential role for type III targeting (31, 37, 38). The observation that LcrV may be surface-displayed together with the result that anti-LcrV antibodies may block type III targeting suggested to us that the assembly of surface-exposed LcrV may be essential for the injection of effector Yop proteins. If so, expression of GST-LcrV in lcrV mutant yersiniae should not restore the targeting of effector Yop proteins. To test this model, HeLa cells were infected with Y. enterocolitica CT1/pVL47 and fractionated with the digitonin technique (Fig. 5). As expected, GST-LcrV was not secreted and localized to the bacterial sediment of the medium and digitonin-extracted HeLa cells (Fig. 5). GST-LcrV induced the expression of the yop virulon. Although large amounts of YopBDEHMNQR were secreted into the extracellular medium, YopEHMN were also found in the supernatant of digitonin-extracted HeLa cells (Fig. 5). Thus, GST-LcrV complemented, at least in part, the targeting defect of lcrV mutant yersiniae, suggesting that intracellular LcrV, but not secreted or surface-exposed LcrV, is required for the type III targeting mechanism.

To examine the type III targeting of HeLa cells by immunofluorescence microscopy, infected tissue cultures were stained with anti-YopE antibodies (Fig. 6). A YopE-specific signal could be detected in the cytosol of HeLa cells that had been infected with wild-type yersiniae. However, infection with the lcrV mutant strain generated a much weaker YopE signal in the cytosol of HeLa cells at a fluorescence intensity level resembling the background level. As a control, HeLa cells that had been infected with the yopE mutant LC2 or the lcrD mutant KUM1 (defective type III machine) also failed to generate a fluorescent signal (data not shown). Y. enterocolitica CT1, expressing LcrV (pVL49) or GST-LcrV (pVL47), generated a YopE-specific immunofluorescent signal in the cytosol of HeLa cells, indicating that LcrV as well as GST-LcrV restored the type III targeting defect of lcrV mutant yersiniae (Fig. 6).

View larger version (128K): [in this window] [in a new window]   Fig. 6.   Immunofluorescence microscopy of HeLa cells infected with Y. enterocolitica strains. Samples were fixed with formaldehyde and stained with anti-YopE antibodies, followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody. Fluorescence and dark-field emission images were captured in an Olympus microscope using a Hamamatsu CCD camera and fused. The YopE signal is indicated in green. YopE was detected in the cytosol of HeLa cells infected with Y. enterocolitica strains W22703/pDA37 (A), CT1/pVL49 (C), and CT1/pVL47 (D). The YopE signal was reduced in HeLa cells infected with the lcrV mutant strain CT1/pDA37 (B). Each experiment was performed using identical light and fluorescence intensity conditions. No attempt was made to optimize the selection of microscopic fields or staining patterns.

Our results suggest that expression of LcrV is essential for the induction of the yop virulon and the targeting of effector Yop proteins during tissue culture infections. We wondered whether overexpression of GST-LcrV as well as LcrV in wild-type yersiniae leads to sequestration of LcrG, thereby prematurely activating the type III pathway. HeLa cells were infected with Y. enterocolitica strain W22703/pVL47 or W22703/pVL49 and analyzed by digitonin fractionation (Fig. 4). Constitutive expression of GST-LcrV and LcrV from the tac promoter caused premature expression of the yop virulon and secretion of YopBDHMR into the extracellular medium (Fig. 4). Nevertheless, type III targeting of yersiniae was not blocked, as YopEHMN were also found in the supernatant of digitonin-extracted HeLa cells (Fig. 4). Thus, overexpression of GST-LcrV or LcrV in a wild-type strain leads to up-regulation of the Yersinia type III pathway, but does not cause the predicted Los phenotype (massive secretion of YopEHMNQ), as observed for lcrV mutant yersiniae expressing GST-LcrV. These results suggest that secreted LcrV acts to ensure specificity of the targeting process.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Following host entry, pathogenic yersiniae are exposed to 37 °C, an environmental condition that triggers the expression of the transcriptional activator LcrF (VirF) and the assembly of the type III machinery (47-49). Although high concentrations of calcium within extracellular fluids prevent complete activation of the type III pathway, we show here that yersiniae secrete YopBDR and LcrV into the extracellular medium during the early stages of infection. YopD and LcrV appear to fulfill a regulatory role (31, 50), and their secretion could provide a measure for yersiniae that the type III machinery is assembled and functional. YopB and YopR, on the other hand, are thought to be dispensable for the regulation of type III pathways, but are necessary for the establishment of acute disease in a mouse model system (9). The role of these virulence factors during the pathogenesis of Yersinia infections has thus far not been characterized. Attachment of yersiniae to host cells appears to transform the type III pathway into an injection device that promotes the type III targeting of YopEHMNOPT into the eukaryotic cytosol.

Intrabacterial LcrV is absolutely required for type III targeting to occur. This requirement could be due to the LcrV·LcrG-mediated regulation of the lcrGVHyopBD operon, encoding another factor (YopD) that is required for type III targeting (9, 11, 14). Although the molecular details of regulation are not known, we would like to entertain two possibilities of altering the ratio between LcrV and LcrG in bacteria. Attachment of yersiniae to host cells may trigger increased expression of LcrV, and the burst of LcrV synthesis could sequester LcrG molecules. As lcrG and lcrV are transcribed from the same promoter, any burst of LcrV expression would have to be regulated at a level of post-transcriptional control. The second model predicts that attachment of yersiniae to eukaryotic cells results in a block of LcrV secretion, raising the intracellular LcrV concentration and sequestering LcrG molecules. Currently, available data do not permit a clear distinction between the two models. It should be noted, however, that even small changes in the intracellular concentration of LcrV could exert a regulatory effect, as they would be accompanied by reciprocal changes of the regulatory molecule LcrG.

Secretion of LcrV during tissue culture infection of yersiniae ensures the specificity of Yop protein injection into eukaryotic cells. We cannot exclude the possibility that LcrV may play a role in the formation of a type III injection device (30, 37). Using immunofluorescence microscopy, discrete patterns of LcrV staining have been revealed on the Yersinia surface (37, 38). Whether this staining is caused by the assembly of LcrV filaments or by the secretion of LcrV into the surrounding medium is not yet resolved (37, 38). In other work, we have found that Y. enterocolitica cells harbor intracellular, soluble LcrV·LcrG complexes, but no membrane-associated LcrV.¹ These data are not consistent with a model whereby LcrV forms a structure that assembles on the Yersinia outer membrane surface. The role of surface-exposed LcrV in the type III targeting mechanism has been examined by several investigators. Some, but not all, experiments suggest that antibodies raised against purified LcrV can block the type III targeting mechanism (37, 38). Nevertheless, LcrV antiserum is known to protect animals against an experimental challenge of Y. pestis (51-54). When injected into mice, purified LcrV reduces tumor necrosis factor- and interferon- production (55), in addition to preventing the chemotaxis of neutrophils (36). Thus, it is conceivable that anti-LcrV antibodies may neutralize the immunosuppressive effects of secreted LcrV molecules rather than interfere with the type III targeting mechanism (38).

Recent observations suggest that LcrG fulfills two functions.¹ Binding of LcrG to the C-terminal domain of LcrV initiates LcrV into the type III secretion pathway.¹ Furthermore, LcrG is required to prevent expression of the yop virulon under non-inducing conditions (34).¹ We think the inability of lcrG mutant yersiniae to secrete LcrV likely causes the observed Los phenotype of this strain.¹ Consistent with this model is our observation that lcrV mutant yersiniae overexpressing GST-LcrV display the same Los phenotype as the lcrG mutant strain. In contrast, wild-type yersiniae expressing GST-LcrV maintain at least some specificity of injection (YopEN), presumably because this strain is still capable of secreting wild-type LcrV.

	ACKNOWLEDGEMENTS

We thank members of our laboratory for critical comments on this manuscript.

	FOOTNOTES

^* This work was supported in part by United States Public Health Service Grant AI 42797.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a fellowship from the National Science Foundation.

^§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095. Tel.: 310-206-0997; Fax: 310-267-0173; E-mail: olafs@ucla.edu.

Published, JBC Papers in Press, August 4, 2000, DOI 10.1074/jbc.M002467200

¹ K. DeBord, V. T. Lee, M. Chu, and O. Schneewind, submitted for publication.

³ D. M. Anderson and O. Schneewind, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GST, glutathione S-transferase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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