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. 2015 Mar 25;10(3):e0120471.
doi: 10.1371/journal.pone.0120471. eCollection 2015.

Random mutagenesis identifies a C-terminal region of YopD important for Yersinia type III secretion function

Rebecca Solomon  1 , Weibing Zhang  1 , Grace McCrann  2 , James B Bliska  3 , Gloria I Viboud  1
Affiliations

Random mutagenesis identifies a C-terminal region of YopD important for Yersinia type III secretion function

Rebecca Solomon et al. PLoS One. .

Erratum in

Abstract

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168-203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB. [corrected] activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Secretion of the yopD mutants.
A. Schematic representation of YopD protein indicating a predicted transmembrane domain (white), a putative coiled-coil region (dark grey) and an amphipathic α-helix domain (black). Overlined are the N-terminal and C-terminal chaperon-binding regions (53–149 and 278–292). Amino acid substitutions that interfere with pore formation, translocation or both are indicated by asterisks. B. Immunoblot showing Yop secretion (bacterial supernatant) and production (bacterial lysate) for each of the YopD mutants expressed in a yopED background and grown at 37°C at low calcium conditions. Monoclonal antibodies for the detection of YopD and YopB and polyclonal antibodies for YopH and LcrV are described in Material and Methods. Anti-rabbit and anti-mouse IR680 or IR800 were used as secondary antibodies. Infrared signal was detected using the Odyssey imaging system (LI-COR Biosience). Quantification of the YopD signal intensities was performed using Odyssey imaging system software. The ratio between the amount of YopD secreted and YopD produced was calculated for each mutant and normalized to wild type YopD.
Fig 2
Fig 2. Effect of the different yopD mutations on translocation.
A. yopD variants were expressed ectopically in a yopD single mutant strain and tested for YopE-mediated cell rounding of HeLa cells. Bacteria cultured under conditions that repress the T3SS were used to infect HeLa cells at MOI of 100 for 1h. YopE-mediated cytotoxicity was assessed by inverted microscopy using a 40× objective. Images were recorded using a digital camera. Images are representative of three different experiments.
Fig 3
Fig 3. Effect of the different YopD mutations on pore formation.
A. HeLa cells were left uninfected or infected with yopEHJD, yopEHJD/pYopD and yopEHJD expressing the different yopD mutants at MOI of 100. After 3 h infection, culture supernatants were removed and tested for LDH release. Background LDH released from uninfected cells was subtracted from infected wells. Results were normalized to yopEHJD/pYopD (100%). Error bars represent the standard deviation of the mean values obtained from four duplicate experiments. ** P<0.0001, * P<0.0005 determined by t-test. B. HeLa cells on coverslips were infected as described above for the LDH assay, and stained with DEAD-LIVE kit as described in Material and Methods. Cells with disrupted membranes exhibit a red nuclei staining.
Fig 4
Fig 4. YopD-dependent activation of proinflammatory signaling.
A. HeLa cells infected with yopEHJD, yopEHJD/pYopD and yopEHJD expressing the different YopD mutants at MOI of 100 for 1h. Cells were washed, lysed with sample buffer 1X, separated by SDS-PAGE and analyzed by immunoblotting with rabbit anti-phospho ERK. Monoclonal antibody against tubulin was used as a loading control. Quantification of the signal intensities was performed using Odyssey imaging system software. Values were normalized to tubulin.
Fig 5
Fig 5. Interaction of the different YopD mutants with YopB.
A. YopB-YopD complexes secreted in bacterial supernatants of yopD, yopD/pYopD and yopD expressing the different yopD mutants were precipitated by 1 h incubation with YopD mab (clone 248:19) followed by incubation with Dynabeads Protein G (Invitrogen). After 3 washes, beads were resuspended in 2X Laemmli sample buffer, and boiled. The eluted material (IP) and an aliquot of the bacterial supernatants (SN) were resolved in SDS-PAGE. Western blot was performed using anti-YopD and anti-YopB Mabs, and anti-mouse IR680 or IR800 secondary antibodies. Bands corresponding to YopB and YopD are indicated by arrows. Also present in the IP samples are a band originated from the beads (marked with an asterix), and a weak band of unknown origin that migrates right below YopB. B. Signal intensities of immunoprecipitated YopB and YopD were calculated using Odyssey imaging system software (LI-COR Biosience). YopB-YopD interaction for each mutant was calculated as the ratio between immunoprecipitated YopB and YopD. Results were normalized to yopD/pYopD.
Fig 6
Fig 6. Phenotype of the Y. pseudotuberculosis expressing yopD mutations in its native location.
HeLa cells were infected with yopEHJ, yopEHJD, and yopEHJD expressing yopDI168T, G196R and A273T in their native location. A. Effector translocation was determined by infecting HeLa cells at a MOI of 100 for 2h. Cells were washed, lysed with 1% Triton X100, and soluble and insoluble fractions were separated by SDS-PAGE and analyzed by immunoblotting with rabbit anti-phospho-GSK-3β (Ser9). Monoclonal antibody against GAPDH and total GSK-3β (Ser9) antibody were used as a loading control for the soluble and insoluble fractions, respectively. Quantification of the signal intensities was performed using Odyssey imaging system software. Results are expressed as the ratio between total ph-ERK signal and GAPDH. B. Pore formation was determined as described in Fig. 4 by analyzing the amount of LDH released from culture supernatants of infected cells. Results were normalized to yopEHJ (100%). Error bars represent the standard deviation of the mean values obtained from three duplicate experiments. * P<0.005 determined by t-test. C. YopD-dependent activation of proinflammatory signaling was performed as described in Fig. 5. MAPK activation was analyzed in cell lysates by immunoblotting using rabbit anti-phospho ERK. A monoclonal antibody against GAPDH was used as a loading control and quantification of the signal intensities was performed using Odyssey imaging system software. Results are expressed as the ratio between total ph-ERK signal and GAPDH.
Fig 7
Fig 7. Residues I168, G196 and A273 are not required for virulence of Y. pseudotuberculosis IP2666 in a mouse model.
Eight-week old C57BL/6J mice were infected orogastrically with 2×109 CFU of wild type Y. pseudotuberculosis IP2666 and its derivative expressing yopD I168T, G196R and A273T. Mouse survival was monitored for 14 days. Results shown are pooled from two independent experiments using 3 mice per group in each experiment. Statistical analysis using log rank test showed no significant differences between the survival curves of each of the mutants and that of the wild type.
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References

    1. Buttner D. Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev. 2012. Jun;76(2):262–310. 10.1128/MMBR.05017-11 - DOI - PMC - PubMed
    1. Viboud GI, Bliska JB. Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu Rev Microbiol. 2005;59:69–89. - PubMed
    1. Sory MP, Cornelis GR. Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol. 1994. Nov;14(3):583–94. - PubMed
    1. Rosqvist R, Magnusson KE, Wolf-Watz H. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 1994. Feb 15;13(4):964–72. - PMC - PubMed
    1. Persson C, Nordfelth R, Holmstrom A, Hakansson S, Rosqvist R, Wolf-Watz H. Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol Microbiol. 1995. Oct;18(1):135–50. - PubMed

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