Previous studies have established a strong correlation between functional T3SS-mediated translocation and the ability to induce pore formation as measured by hemolytic activity in infected erythrocytes (Håkansson et al., 1996; Holmstrom et al., 1997; Neyt and Cornelis, 1999; Olsson et al., 2004; Ryndak et al., 2005). When the LcrV+1 and LcrV−1 strains were used to infect erythrocytes they were essentially unable to induce any significant hemolytic activity (Figure 1). In fact, the levels of hemolysis were found to be similar to that induced by ΔlcrV, ΔyopB, or ΔyopD mutants. Of note, the analysis was performed in a ΔyopK mutant background as the levels of both hemolysis and translocation, are known to be highly up-regulated in the absence of YopK (Holmstrom et al., 1997). These results indicate that the two LcrV+/−1 strains are unable to induce pore formation. Since functional LcrV is required for membrane insertion of YopB and YopD (Broz et al., 2007), we therefore wanted to establish if these mutants could still localize YopB and YopD to the cell membrane or not. To this end, membranes from infected erythrocytes were isolated by floatation on a sucrose gradient and subjected to SDS-PAGE and Western blot analysis using YopB and YopD antisera. By this approach, we were unable to detect YopB, and the levels of YopD were very low, in the membranes of cells infected with the two LcrV+/−1 strains both in parental background and in the ΔyopK mutant background (Figure 2). As expected (Thorslund et al., 2011), infection with the ΔyopK mutant resulted in increased levels of both YopB and YopD in the membranes (Figure 2). Control experiments verified that during infection the total levels of YopB and YopD were similar for all strains except for the ΔlcrV mutant, for which the expression of the T3SS encoding genes is known to be down-regulated (Pettersson et al., 1999) (Figure S2). Overall, the phenotype of the LcrV+/−1 strains was similar to a ΔlcrV mutant with respect to hemolytic activity and membrane insertion of YopB and YopD. Thus, the LcrV+/−1 strains evidently induced YopE mediated cytotoxicity, as reported previously (Bröms et al., 2007), without localizing YopB and YopD to the erythrocyte membrane.

As mentioned above, past studies have shown a strong correlation between lytic activity due to pore formation and the functional translocation of T3SS effectors (Håkansson et al., 1996; Holmstrom et al., 1997; Neyt and Cornelis, 1999; Olsson et al., 2004; Ryndak et al., 2005). Hence, it was important to verify that the LcrV+/−1 mutants that lacked hemolytic activity really had retained translocation ability. In our previous study we found that these mutants, similar to the wild type strain, induced rapid cytotoxicity in infected HeLa cells (Bröms et al., 2007). In this assay very low levels of translocated YopE are needed for visible cell rounding. Therefore, to detect any possible translocation deficiency, we repeated the assay using different MOIs. At a high MOI (40:1) the wild type strain induced full cytotoxicity within an hour of infection, while at a low MOI (2.5:1), full cytotoxicity was delayed until 3–4 h of infection. Using this approach it was clear that the LcrV+/−1 strains induced a cytotoxic response with similar kinetics as the wild type strain (data not shown), even at MOI 2.5. Since cytotoxic response is an indirect measurement of intracellular targeting of YopE, we decided to confirm more directly that YopE was translocated by the LcrV+/−1 strains. To this end, infected HeLa cells were fixed, permeabilized and incubated with rabbit-anti-YopE antisera followed by analysis using laser scanning confocal microscopy. YopE specific staining was detected in cells infected with the LcrV+/−1 mutants as well as in cells infected with the wild type, thereby confirming the ability of the mutants to translocate YopE (Figure 3A). Expectantly, no YopE specific staining was observed in cells infected with the ΔlcrV mutant. To confirm that this translocation was general and not just specific for YopE, we also monitored translocation of YopH using a beta-lactamase reporter system. HeLa cells were infected with Yersinia variants expressing a YopH-Bla fusion protein. Already after 40 min of infection, translocation of the fusion protein (as visualized by the blue fluorescence) was observed in a majority of cells infected with either the wild type or the LcrV+/−1 mutants (Figure 3B), once again confirming the general ability of the LcrV+/−1 mutants to translocate Yop effectors into the host cell. Quantification of the translocated effectors showed that the LcrV+/−1 mutants translocated YopE and YopH at wild type levels (Figure 4).

As previously mentioned, translocation is up-regulated in absence of YopK, which correlates with larger pores being formed (Holmstrom et al., 1997). This prompted us to investigate translocation of Yop effectors by the LcrV+1 and LcrV−1 strains in the ΔyopK mutant background, for which no pore formation was seen (Figure 1). To address this, HeLa cells infected with the different Yersinia strains were fractionated, using a well-established translocation assay (Nordfelth and Wolf-Watz, 2001). The cytosolic proteins were subjected to SDS-PAGE and Western blot using monospecific YopE and YopH antisera. As expected, the ΔyopK mutant translocated increased levels of both YopE and YopH compared to the isogenic wild type strain (Figure 4). The LcrV+1 and LcrV−1 strains also translocated increased levels of YopE and YopH in absence of YopK, at the same level as the ΔyopK mutant (Figure 4). No intracellular YopE and YopH could be detected in cells infected with the ΔlcrV mutant.

Since translocation of effectors occurred although little or no YopB and YopD could be detected in the erythrocyte membranes it was important to establish that effector translocation by the two LcrV+/−1 strains still required YopB and YopD. To address this, the LcrV+/−1 strains were also mutated for yopB and yopD. None of these strains were able to induce cytotoxicity of infected HeLa cells (data not shown) confirming that the effector translocation observed herein still required functional YopB and YopD.

It is puzzling that translocation in the LcrV+/−1 strains is regulated as in the wild type despite the lack of lytic activity. Contrasting with numerous previous studies, our results show that neither lytic activity nor translocator insertion in erythrocyte membranes have any correlation with levels of Yop effector translocation.

Given the observance of translocation in the absence of lytic activity, we decided to examine membrane localization of YopB and YopD in HeLa cells to be consistent with the experimental setup used in the translocation assays described above. We found that the levels of YopB were too low to be detected in cell membranes, regardless of the infecting strain (data not shown). The levels of YopD were very low, yet detectable by Western blot, in HeLa cells infected with the wild type strain (Figure 5). As expected, levels of membrane localized YopD were higher in cells infected with the ΔyopK mutant background and, most importantly, when the two LcrV+/−1 strains were analyzed in this background both mutants were found to insert similar levels of YopD into the membranes as the ΔyopK mutant strain (Figure 5). These findings are very important because above all they verify that using the erythrocyte model to study T3SS mediated pore formation and membrane insertion has limitations and can even give contradictory results that mislead conclusions regarding the T3SS mechanism. While the erythrocyte model is a convenient method, the relevance can be questioned, as erythrocytes are not a natural target cell of Yersinia during infection of an animal host.

With the exception of the experiments involving the erythrocyte infection model, the LcrV+/−1 strains displayed in vitro phenotypes that were very similar to the parental strain. Based on these observations, we anticipated that these mutants would be fully virulent. In order to examine this, we decided to use assays that could discriminate the impact of these mutations on Yersinia virulence.

One major virulence mechanism mediated by the T3SS of Yersinia is the ability to block phagocytosis by professional phagocytes (Rosqvist et al., 1988). To analyze if the LcrV+1 and LcrV−1 strains were affected in the ability to block phagocytosis we infected J774 macrophages with the different Yersinia strains for 30 min. The cells were then fixed and a double staining assay was performed to distinguish between extracellular and internalized bacteria. As expected, the wild type strain efficiently blocked phagocytosis with only 24% of infecting bacteria internalized (Figure 6). In contrast, 80% of the infecting ΔlcrV mutant and 85% of the virulence plasmid cured strain were phagocytosed (Figure 6). Interestingly, the LcrV+1 and LcrV−1 strains showed an intermediary phenotype where 41 and 42% of the bacteria were phagocytosed, respectively (Figure 6). Thus, even though the LcrV+/−1 strains were able to translocate both YopE and YopH to levels similar as the wild type strain, some critical aspect of this translocation process must be compromised in a manner that causes a significant reduction in phagocytosis inhibition.

Previous work has established that early targeting of YopH to focal adhesion complexes is essential for phagocytosis inhibition (Andersson et al., 1996; Persson et al., 1997, 1999). One of the YopH targets is Focal Adhesion Kinase (FAK) and it was shown that the initial phosphorylation of FAK in response to Yersinia binding to the host cell is quickly reversed by the powerful phosphatase activity of YopH. By extension, this deactivation of FAK in turn impairs bacterial uptake (Persson et al., 1997). Hence, in order to establish if the N-terminal mutations in LcrV compromised early YopH targeting, we infected HeLa cells for 10 min and then immunoprecipitated FAK. The phosphorylation level of FAK was analyzed by Western blot, using phospotyrosine antibodies. In cells infected with the YopHC403A strain, FAK remained phosphorylated as this YopH variant is inactive and cannot dephosphorylate its target proteins. In contrast, in cells infected with the wild type strain, only low levels of phosphorylated FAK were detected (Figure 7). Curiously, the LcrV−1 strain also proved to be less efficient in blocking the phosphorylation of FAK compared to the wild type strain. Quantification of the levels of phosphorylated FAK from three independent experiments indicated a 2.13-fold reduction of phosphorylated FAK in cells infected with the wild type strain compared to the YopHC403A strain and a 1.54-fold reduction when compared to the LcrV−1 strain. Similar results were also observed with the LcrV+1 strain (data not shown). Thus, the LcrV+/−1 strains are defective in early targeting of YopH and this in turn impairs their ability to block phagocytosis (Figure 6). These are pertinent findings as they support the notion that LcrV both facilitates YopB/D localization to the host cell membrane and that the N-terminal region of LcrV mediates efficient early delivery of YopH to its molecular targets inside the infected host cell.

Since the LcrV+1 and LcrV–1 strains were impaired in anti-phagocytosis it was necessary to investigate if this correlated to attenuation in the in vivo mouse infection model. To this end, C57/BL6 mice were infected intraperitoneally with the various Yersinia strains. As expected the ΔlcrV mutant was completely attenuated, and all mice survived a high infection dose (1.6 × 10⁶ CFU) without displaying any signs of disease (fuzzy fur, diarrhea, weight loss, listlessness). Mice infected with the wild type strain showed disease symptoms within a few days even at the lowest dose used (2.1 × 10³ CFU) and all mice had succumbed to the infection by day 8 post infection. Strikingly, the LcrV+1 and LcrV−1 strains were essentially as attenuated as the ΔlcrV mutant. All but one mouse survived infection with the high dose of either LcrV+1 or LcrV−1 (Figure 8). Thus, the LcrV+1 and LcrV−1 strains were indeed highly attenuated for virulence, reflecting that a defect in early targeting of YopH has a dramatic impact on in vivo virulence.

The bacterial strains and plasmids used in this study are presented in Table 1. Bacteria were routinely grown at 26°C in Luria Bertani (LB) agar or LB broth supplemented with 1 mM CaCl₂ and 75 mM NaCl. For induction of the T3SS, bacteria were grown at 37°C in secretion permissive medium (5 mM EGTA and 20 mM MgCl₂) (LB–Ca²⁺) unless otherwise stated. Where appropriate the following antibiotics were added to the final concentrations; kanamycin 30 μg/ml, chloramphenicol 25 μg/ml.

HeLa cells were cultivated in Minimum essential medium (MEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma), 2 mM glutamine, 0.035% sodium bicarbonate and 100 IU penicillin. J774.1 cells were cultivated in Dulbecco's modified eagle medium (DMEM glutaMAX) (Gibco) supplemented with 10% FBS and 100 IU penicillin. The cells were kept at 37°C with 5% CO₂. For infection assays cells were seeded in plates 1 day before experiment and 0.5 hour before infection the cells were washed and incubated in non-supplemented MEM or DMEM.

E. coli S17-1 λpir was used to conjugate the suicide plasmids pJEB368 and pJEB369 into YPIII/pIB155 (ΔyopK) to generate ΔyopK/LcrV+1 and ΔyopK/LcrV−1 mutants. ΔyopB and ΔyopD double mutants were constructed by conjugating the suicide plasmids pMF024 and pMF463 into YPIIIpIB10201 (LcrV+1) and YPIIIpIB10202 (LcrV−1). The YopH-Bla fusion was constructed by overlap PCR using the primers listed in Table S1, yielding a DNA fragment corresponding to codons 6–468 of YopH fused to beta-lactamase codons 24–286 (Akopyan et al., 2011). The PCR fragment was cloned into the pcr2.1 TOPO vector (Invitrogen) for amplification, and then sub-cloned into the pNQ705 suicide vector (Milton et al., 1992). S17-1 λpir was used as the donor strain in conjugation with Yersinia pseudotuberculosis wild type (YPIII pIB102), LcrV+1 (YPIII pIB10201) and LcrV−1 (YPIII pIB10202) strains and single recombination of the pNQ-YopH_FL-Bla inactivated the wild type copy of YopH.

Sheep erythrocytes were washed and resuspended in LB–Ca²⁺ Supplemented with 75 mM NaCl and Complete Mini protease inhibitor cocktail (PIC) (Roche). A total of 5 × 10⁹ induced Yersinia bacteria were harvested and resuspended in 5 ml erythrocytes suspension (10¹¹ cells) and centrifuged to create contact between bacteria and cells before incubation at 37°C. Samples were repeatedly resuspended and centrifuged during infection to increase contact between bacteria and cells. After 2 h 100 μL samples were taken and mixed 1:1 with PBS and centrifuged at 3500 g, 5 min, after which 100 μl of the supernatant was transferred to a flat bottom plate and the absorbance at 570 nm was measured to determine the hemolysis.

To analyze total levels of YopB and YopD during infection, 500 mL samples were collected, mixed 1:1 with MQ H₂O and vortexed to lyse the cells. The samples were treated with DNAseI (Thermo Fisher Scientific) according to the manufactures instructions. The samples were centrifuged at 12,000 g, 5 min and the supernatants were collected and subjected to SDS-PAGE and Western blot using an antisera which recognizes both YopB and YopD (ASTI).

To isolate the erythrocytes membranes, 35 ml ice-cold lysis buffer (5 mM Tris-HCl, PIC, pH 7.5) was added to the remaining reactions followed by shaking and incubation on ice 10 min, then 4.5 ml 10X TBS (200 mM Tris-HCl, 1.5 M NaCl, pH 7.5) was added to restore the pH and salt concentration and the samples were centrifuged at 3500 g, 20 min. The supernatants were transferred to thick-wall centrifugation tubes (Beckman) and spun at 100,000 g for 2 h at 4°C. The pellet was resuspended in 50% sucrose in TBS (20 mM Tris-HCl, 150 mM NaCl, PIC, pH 7.5) and sonicated. A step-wise gradient was established in ultra-clear centrifugation tubes by adding the resuspended sample on top of 2 ml 65% sucrose-TBS and overlay with 15 ml 44% sucrose-TBS followed by 5 ml 25% sucrose-TBS. Gradients were centrifuged at 15,000 g for 16 h at 4°C using a Sw-28 rotor (Beckman). The membranes were isolated from the 25/44% interface and washed with TBS before resuspension in 150 μl TBS. The total protein concentration was estimated in a NanoDrop spectrophotometer at A₂₈₀ and 20 μg was loaded on a 12% SDS-PAGE and analyzed by Western blot using YopD polyclonal antisera and YopB monoclonal antibody.

A total of 10⁵ HeLa cells were infected with induced Yersinia strains at MOI ranging from 2.5:1 to 40:1 and the level of cytotoxicity was observed in an inverted light microscope, repeatedly during up to 4 h post infection. For immunostaining a MOI of 25:1 was used. The cells were washed and fixed in 2% paraformaldehyde after 1.5 h infection. The cell membranes were stained with TexasRed conjugated wheat germ agglutinin (Molecular Probes, Invitrogen) before they were permeabilised in TSB (0.5% Triton X-100 in a buffer consisting of 4% PEG 6000, 1 mM EGTA and 100 mM Pipes pH 6.8). Intracellular YopE was detected using an affinity purified polyclonal YopE antibody followed by an Alexa488-conjugated secondary antibody (Molecular probes, Invitrogen). The nucleus and bacteria were stained with DAPI. Images were taken with laser scanning confocal microscopy (Leica).

YopH translocation was analyzed using a beta-lactamase reporter system (Charpentier and Oswald, 2004; Marketon et al., 2005) where full length YopH was fused to beta-lactamase. HeLa cells were seeded in 35 mm glass bottom dishes (MatTek) 1 day before the experiment. Prior to infection the cells were labeled with the FRET substrate CCF4-AM (Invitrogen) according to the manufactures instructions. Induced Yersinia strains were added to the cells at a MOI of 50:1 and incubated 40 min at room temperature. Images were taken with a live cell microscope (Nikon Eclipse Ti-E), equipped with a true color camera, using a long pass filter to detect the two wave length of the FRET substrate.

The experiment was performed essentially as described before (Nordfelth and Wolf-Watz, 2001). In short, 5 × 10⁶ HeLa cells were infected with induced Yersinia strains at a MOI of 50:1 in the presence of Cytochalasin D (0.5 μg/ml). After infection the cells were washed in PBS and treated with 0.5 mg/ml Proteinase K (Roche Diagnostics, Gmbh) for 1 min. The cells were aspirated and incubated 20 min at room temperature, after which, 4 mM PMSF was added to inactivate remaining Proteinase K. The cells were lysed with 0.4% Digitonin in a total volume of 1 ml. Cell debris and bacteria were pelleted by centrifugation at 15,000 g, 10 min, 4°C. The supernatant was separated on a 12% SDS-PAGE and analyzed by Western blot using affinity purified antisera against YopE and YopH or monoclonal Tubulin antibody (Sigma Aldrich). The protein levels were quantified by use of the LAS4000 image reader and Multi Gauge-Image software (Fujifilm). The levels of YopE and YopH were normalized against Tubulin levels. The results were analyzed using the paired Student's t-test with the significance set at p ≤ 0.05^*, p ≤ 0.01^** and p ≤ 0.001^***.

A total of 10⁷ HeLa cells were infected in duplicates with Yersinia strains at a MOI of 50:1 in 15 cm dishes. After 1 h at 37°C the cells were put on ice for 5 min and washed twice with ice cold PBS. The cells were scraped off in 5 mL ice-cold PBS and suspension from the duplicate dishes were pooled in a centrifugation tube. The cells were pelleted at 290 g, 5 min, resuspended in 1 mM EGTA, 1 mM MgCl₂, 1 mM DTT, and incubated on ice for 8 min after which the cells were pelleted again at 290 g, 5 min. The cells were resuspended in 0.3 mL 250 mM sucrose, 3 mM Imidazole and Complete Mini protease inhibitor cocktail (Roche) (total volume ~0.4 mL) and lysed by passing 40 times through a 23 G needle. The lysate was centrifuged at 900 g, 5 min and the supernatant was collected. The pellet was washed with 1 mL sucrose buffer and spun again at 900 g and the supernatant was pooled to the previous one and the samples were spun at 14000 g, 5 min. The resulting supernatant was centrifuged at 125000 g for 1 h using a Ti70.1 rotor (Beckman). The pellet containing the cell membranes was resuspended in 100 μL 2X SDS loading buffer. Equal amounts of protein were loaded on a 12% SDS-gel and analyzed with Western blot using YopD polyclonal antisera. The levels of YopD were quantified using the Multi Gauge-Image software (Fujifilm) and normalized to the unspecific band. The results were analyzed using the paired Student's t-test with the significance set at p ≤ 0.05^*, p ≤ 0.01^** and p ≤ 0.001^***. NS, No significant difference.

A total of 10⁵ J774a.1 cells were infected with Yersinia strains at a MOI of 20:1. After 30 min infection unattached bacteria were washed away with PBS and the cells were fixed in 0.4% paraformaldehyde. Extracellular bacteria were stained using Yersinia antisera followed by Alexa568-conjugated antibody (Molecular Probes, Invitrogen). The cells were permeabilised with 0.5% Triton X-100 and both extra- and intracellular bacteria were stained with Yersinia antisera followed by Alexa488-conjugated antibody (Molecular Probes, Invitrogen). Samples were viewed in a fluorescence microscope and the total amount of cell associated bacteria and extracellular bacteria were counted manually. The results were analyzed using the Wilcoxon signed-rank test. Each experiment was analyzed separately and significance was set at p ≤ 0.05^*, p ≤ 0.01^** and p ≤ 0.001^***.

A total of 4 × 10⁶ HeLa cells were infected with Yersinia strains at a MOI of 100:1 in a small volume to create instant contact between bacteria and cells. The infection was terminated at different time points by washing two times with ice-cold PBS + 0.1 mM Na₃VO₄. The cells were lysed in ice-cold precipitation buffer [50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1% Nonidet NP-40, 0.25% sodium deoxycholate, 1 mM Na₃VO₄ and Complete Mini protease inhibitor cocktail (Roche Diagnostics, Gmbh)] at 4°C for 20 min after which the cells were scraped off and centrifuged at 16000 g for 10 min. The lysate was pre-incubated with mouse IgG coated protein G Sepharose beads (4 Fast Flow, Amersham Biosciences, Sweden) at 4°C for 1 h. The beads were spun down and the pre cleared lysate was incubated with anti-FAK (clone 2A7, Upstate Biotechnology, Lake Placid, NY) coated proteinG sepharose beads at 4°C for 3 h. The beads were washed two times in precipitation buffer and the bound material was eluted in a small volume of 2X SDS loading buffer at 95°C for 5 min. Samples were subjected to SDS-PAGE and the amount of phosphorylated FAK was analyzed by Western Blot using a phosphotyrosine antibody (Clone 4G10) (Millipore). Images were acquired in a LAS4000 image reader and the signal intensity was quantified using Multi Gauge-Image software (Fujifilm). The results were analyzed using the paired Student's t-test with the significance set at p ≤ 0.05^*, p ≤ 0.01^** and p ≤ 0.001^***. NS, No significant difference.

Bacteria were grown overnight in unsupplemented LB at room temperature and harvested and diluted in PBS to final concentrations ranging from 10⁴to 10⁶ bacteria/ml for the wild type and LcrV frameshift mutants. The attenuated ΔlcrV mutant was used as a negative control at concentrations 10⁵–10⁷ bacteria/ml. Five C57BL/6 mice were infected intraperitoneally with 100 μl for each strain and dilution. Bacteria were plated on LA plates in parallel to determine the exact infection dose. The experiments were conducted in accordance with the ethical permission and all mice were monitored for symptoms twice daily and mice showing severe symptoms were sacrificed. Based on our previous experience mice with severe symptoms normally succumb to the infection within 24 h. The animal experiment was approved by the local animal ethics committee at Umea University (Dnr A144-12).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.