Vibrio cholerae MARTX toxin heterologous translocation of beta-lactamase and roles of individual effector domains on cytoskeleton dynamics

Abstract

The Vibrio cholerae MARTXVc toxin delivers three effector domains to eukaryotic cells. To study toxin delivery and function of individual domains, the rtxA gene was modified to encode toxin with an in-frame beta-lactamase (Bla) fusion. The hybrid RtxA::Bla toxin was Type I secreted from bacteria; and then Bla was translocated into eukaryotic cells and delivered by autoprocessing, demonstrating that the MARTXVc toxin is capable of heterologous protein transfer. Strains that produce hybrid RtxA::Bla toxins that carry one effector domain in addition to Bla were found to more efficiently translocate Bla. In cell biological assays, the actin cross-linking domain (ACD) and Rho-inactivation domain (RID) are found to cross-link actin and inactivate RhoA, respectively, when other effector domains are absent, with toxin autoprocessing required for high efficiency. The previously unstudied alpha-beta hydrolase domain (ABH) is shown here to activate CDC42, although the effect is ameliorated when RID is also present. Despite all effector domains acting on cytoskeleton assembly, the ACD was sufficient to rapidly inhibit macrophage phagocytosis. Both the ACD and RID independently disrupted polarized epithelial tight junction integrity. The sufficiency of ACD but strong selection for retention of RID and ABH suggests these two domains may primarily function by modulating cell signaling.

Fig. 1. Schematic representation of MARTX toxins expressed by V. cholerae strains generated for this study

Domain organization of each toxin is shown to scale with grey-hatched bars representing the locations of the MARTX repeat regions. Effector domains are: the actin cross-linking domain (ACD, red), the Rho-inactivation domain (RID, green), and the alpha-beta hydrolase domain (ABH, purple). The location of surrogate effector domains beta-lactamase (Bla without secretion signal) is shown in blue. The cysteine protease domain (CPD) required for autoprocessing to release effector domains and/or Bla to eukaryotic cells after translocation is depicted in light blue. The relative position of catalytic residues for each effector domain and the CPD are shown numbered according to the full-length 4545 aa RtxA protein as annotated by Lin et al. (1999) and verified by Dolores & Satchell (2013). Key at right indicates V. cholerae strain designations for rtxA arrangements in isogenic ΔhapAΔhlyA background that is defined as wild type (WT) in this study. Those that carry the C3568A mutation in CPD for non-processing (NP) or catalytically inactivating (CIA) mutations as indicated in the diagram. Detailed genetics of strains are listed in Table 1.

Fig. 2. RtxA::Bla strains are ampicillin resistant but do not induce HeLa cell lysis

(A) A BBL Sensi-Disc (ampicillin, AM10) was placed in the center of an LB agar plate evenly spread with V. cholerae strain as indicated. Plates were photographed after overnight incubation at 37°C and the area of the zone of clearance was calculated according to A=πr². V. cholerae strains used (in order shown) are KFV119 (wild-type), JD1 (rtxA::bla), JD5 (rtxA::bla C3658A), JD4 (rtxA::bla rtxB::km). Statistical significance compared to KFV119 is indicated as determined by multiple comparisons after one-way ANOVA (**p<0.005)

Fig. 3. Translocation of Bla to HeLa cells is more efficient when an effector domain is present

Translocation of Bla activity was determined using CCF2 fluorescence from V. cholerae treated cells quantified by flow cytometry. (A,B) Number of cells and percentage of cells from 10,000 events were determined using the AmCyan emission channel for CCF2⁺ cells (green peaks, upper panels) and then gated for those that also shifted to positive for emission in the Pacific Blue channel for Bla⁺ cells (blue peaks). The threshold for assignment as CCF2⁺ was determined by gating according to unstained, untreated cells (grey peaks) and for Bla⁺ cells by gates set according to mock (PBS) treated cells (second set of panels from right in row A. Strains used are as indicated in figure. (C) Percent Bla⁺ cells for additional samples quantified by the same method are shown as histograms alongside data from panels A and B. Additional strains for this panel are non-processing strains JD22, JD21, and JD15 and catalytically inactive JD24, JD25, and SAG1 that produce carry variant forms of the toxin gene as shown in the legend and described in Fig. 1 and Table 1. Data shown are the average and standard deviation of two biological replicates. Statistical significance in panel C was determined by multiple comparisons after one-way ANOVA (**p<0.005 and ***p<0.001 compared to unmodified rtxA::bla; ^†p<0.05 compared to paired sample without mutation; ns is not significant compared to paired sample without mutation). na indicates not applicable.

Fig. 4. The ACD is sufficient for actin cross-linking and requires CPD autoprocessing

Proteins in cell lysates prepared from HeLa cells treated with V. cholerae or mock-treated with PBS for 90 min were separated by 8% SDS-PAGE and actin visualized by western blotting with anti-actin antibody. Detection of tubulin is shown as a loading control. Monomeric (Mo) and cross-linked dimers (Di), trimers (Tr), and tetramers (Te) are indicated. V. cholerae strains used (in order shown) are KFV119 (wild-type), JD23 (ΔrtxABCD), JD1 (rtxA::bla), JD20 (rtxA::acd-bla), JD22 (acd-bla C3568A), and JD24 (acd-bla E1990A). Experiment shown is representative of two independent experiments.

Fig. 5. Reduction of Rho-GTP by RID is only partially dependent on autoprocessing

Active RhoA in cell lysates prepared from HeLa cells treated with V. cholerae or mock-treated with PBS for 4 h was measured by G-LISA. Absorbance data are normalized as percent difference compared to the average absorbance in each experiment for cells treated with rtxA null strain JD23 to limit data to effects due to the toxin and not due solely to exposure of cells to bacteria. Upper panel shows total RhoA and actin loading control in cell lysates from a representative experiment by western blotting. V. cholerae strains used (in order shown) are JD23 (ΔrtxABCD), CCO5 (rtxAΔacd), BGV1 (rtxAΔacd C3022A), JD1 (rtxA::bla), JD19 (rtxA::rid-bla), JD25 (rid-bla H2782A), and JD21 (rid-bla C3568A). Three to nine pooled data points are from three experiments performed with three biological replicates. Statistical significance between samples indicated as determined by multiple comparisons after one-way ANOVA (**p<0.005, ***P<0.001).

Fig. 6. The ABH domain activates CDC42 dependent upon residues C3568 and H3369

Active CDC42 in cell lysates prepared from HeLa cells treated with V. cholerae or mock-treated with PBS for 4 h was measured by G-LISA™. Absorbance data are normalized as percent difference compared to the average absorbance in each experiment for cells treated with rtxA null strain JD23 to limit data to effects due to the toxin and not due solely to exposure of cells to bacteria. Upper panel shows total CDC42 in the same cell lysates by western blotting. V. cholerae strains used (in order shown) are JD23 (ΔrtxABCD), CCO5 (rtxAΔacd), BGV1 (rtxAΔacd C3022A), JD1 (rtxA::bla), JD2 (rtxA::abh-bla), Sag1 (abh-bla H3369A) JD15 (abh-bla C3568A), and JD19 (rtxA::rid-bla). Three to nine pooled data points are from three experiments performed with three biological replicates. Statistical significance between samples indicated as determined by multiple comparisons after one-way ANOVA (**p<0.005, ***P<0.001; ^†p<0.001 compared to all other samples except CCO5)

Fig. 7. ACD is sufficient to inhibit phagocytosis by J774 macrophages

Cultured J774 macrophage cells were exposed to V. cholerae strain for 45 min and then assayed. (A) Percent cell lysis was determined as described in experimental procedures. (B) Translocation of Bla was quantified as shown in Fig. 3 with percent CCF2+/Bla+ cells from 10,000 cells shown as a histogram. (C) Percent phagocytosis was determined by uptake of fluorescent pHrodo Green E. coli Bioparticles. V. cholerae strains used in panel A (in order shown) are KFV119 (wild-type), JD23 (ΔrtxABCD), JD1 (rtxA::bla), JD20 (rtxA::acd-bla), JD19 (rtxA::rid-bla), and JD2 (rtxA::abh-bla). Additional strains in panels B and C are JD4 (rtxA::bla rtxB::km), JD5 (rtxA::bla C3568A), and JD22 (rtxA::acd-bla C3568A). Data shown in panels A and C are the average and standard deviation of three biological replicates while panel B represents biological duplicates. Statistical significance between samples indicated as determined by multiple comparisons after one-way ANOVA (**p<0.05 and ***P<0.005 are compared to rtxA::bla. ^†p<0.05 compared to paired sample without mutation)

Fig. 8. Both ACD and RID independently disrupt tight junctions in the polarized T84 monolayers

Polarized T84 colonic carcinoma cells were cultured to resistance ~1000 Ω cm². V. cholerae was added to the apical side of the monolayer and resistance across the monolayer was measured every 10 min for a period of 250 min. Genotype of V. cholerae strains used are indicated in legends below panels and correspond to strains (in order A–D) KFV119 (wild type), JD23 (ΔrtxABCD), JD1 (rtxA::bla), JD5 (rtxA::bla C3568A), JD20 (rtxA::acd-bla), JD19 (rtxA::rid-bla), JD2 (rtxA::abh-bla), JD24 (rtxA::acd-bla E1990A), and JD21 (rtxA::rid-bla C3568A). Data shown are the average and standard deviation of three biological replicates and representative of at least two experiments for each of the strains. Symbol § designates the time point at which the percent sample initial resistance became statistically significant (p<0.05) from either the JD23 control (panels A and C) or the paired sample with a catalytically inactive mutation (panel D).