Rejection of impassable substrates by Yersinia type III secretion machines

Abstract

Type III machines of pathogenic Yersinia spp. transport Yop proteins across the bacterial envelope into host cells. Translational fusions of yopE to the dihydrofolate reductase gene (dhfr) or the beta-galactosidase gene (lacZ) generate hybrid proteins that block type III injection of Yop proteins into host cells, consistent with the canonical view that impassable DHFR and LacZ hybrids jam secretion machines. Mutations in repressors of posttranscriptional gene regulation, Yersinia enterocolitica yscM1 and yscM2 as well as Yersinia pestis lcrQ, relieve the YopE-DHFR-imposed blockade and restore type III injection into host cells. Genetic suppression of the type III blockade does not, however, promote YopE-DHFR secretion. A model is proposed whereby rejection of YopE-DHFR from the secretion pathway inhibits type III gene expression.

FIG. 1.

Effect of low-copy-number (pVL20) and medium-copy-number (pMAF35) plasmids carrying yopE-dhfr on type III secretion of Y. enterocolitica W22703. Type III secretion was measured by separating the medium supernatant (S) and the bacterial pellet (P) of centrifuged cultures that had been induced by temperature shift and low calcium (+ or −). (A) Proteins in both fractions were separated by SDS-PAGE and stained with Coomassie brilliant blue. The positions of molecular weight markers are indicated. (B) Proteins were also electroblotted onto a PVDF membrane and detected with specific antisera. α, anti.

FIG. 2.

Plasmids carrying yopE-dhfr (pJS93) or yopE-lacZ (pJS94), but not the vector control (pJS92), block type III secretion of Y. pestis KIM8. (A) Coomassie brilliant blue-stained, SDS-PAGE-separated proteins in the Yersinia medium supernatant (S) and the bacterial pellet (P). (B) Immunoblot analysis of protein localization in the medium supernatant and the bacterial pellet. α, anti. (C) Quantification of immunoblot analysis in panel B for the percent amount of secreted protein and comparison between bacteria harboring pJS92 (vector control) and pJS93 (yopE-dhfr). Bars represent the means of three independent experiments. The error bars represent the standard errors of the means. Significance was determined using Student's t test. (D) Total amounts of Yersinia proteins were determined by immunoblotting of proteins from whole-culture samples and compared between bacteria expressing YopE-DHFR (pJS93) and the vector control (pJS92).

FIG. 3.

YopE-DHFR blocks the Y. pestis LCR. Y. pestis strain KIM8 was transformed with a vector control plasmid or with the same vector expressing yopE-dhfr from the IPTG-inducible tac promoter (pJS95). Strains were streaked on HIA plates and incubated at 37°C with or without calcium, the latter of which is an inducer of the type III pathway, and with or without IPTG, an inducer of the tac promoter.

FIG. 4.

YopE-DHFR blocks Y. pestis type III injection into HeLa cells. (A) Y. pestis strain KIM8(pJS95) was inoculated into HeLa cell monolayers for 3 h at 37°C. Infected tissue cultures were fractionated as described in Materials and Methods, and protein localization was analyzed by immunoblotting with specific antisera. M, medium fraction; D, digitonin fraction; S, supernatant fraction; P, pellet fraction; α, anti. (B) Data in panel A were analyzed to determine the percent amount of injected polypeptide. The bars indicate the means of three independent experiments. The error bars indicate 1 standard deviation of the mean. Significance was calculated using Student's t test (*, P < 0.01; **, P < 0.05). (C) Y. pestis cytotoxicity for HeLa cells was measured by staining F-actin with Texas red-conjugated phalloidin. Differential interference contrast (DIC) images were captured at a magnification of ×600, and fluorescence was measured at 608-nm emission. Samples were compared with a slide that received no bacteria (uninfected). (D) Model for YopE-DHFR-mediated block of type III injection.

FIG. 5.

YopE-DHFR blocks Y. enterocolitica type III injection into HeLa cells. (A) Y. enterocolitica strain W22703(pJS95) was inoculated into HeLa cell monolayers for 3 h at 37°C and subjected to digitonin fractionation as described in Materials and Methods. α, anti. (B) Data in panel A were analyzed to determine the percent amount of injected polypeptide.

FIG. 6.

Mutations in class II genes relieve the type III blockade of YopE-DHFR. Y. enterocolitica W22703(pJS92) (vector control) and strains W22703, VTL1 (ΔyopN), MC2 (ΔlcrG), VTL2 (ΔyopD), CT133 (ΔlcrH), and EC2 [Δ(yscM1 yscM2)], each carrying pJS93, were analyzed for type III secretion in the absence of calcium. (A) Coomassie brilliant blue-stained SDS-PAGE gel for separation of proteins in the medium supernatant (S) and the bacterial pellet (P). The positions of molecular weight markers are indicated. (B) Proteins in the supernatant and bacterial pellet fractions were also electroblotted onto a PVDF membrane, analyzed with specific antisera for strains W22703 (wild type) and VTL2 (ΔyopD), and quantified for the percent amount of secreted polypeptide. (C) Total amounts of Yersinia proteins were determined by immunoblotting proteins from whole-culture samples and compared between bacteria expressing YopE-DHFR (pJS93) and the vector control (pJS92).

FIG. 7.

Expression of yopE-dhfr does not affect yop transcript levels but results in an inhibition of Yop protein synthesis. (A) Total RNA was isolated from Y. enterocolitica W22703(pJS95) that had been induced for type III secretion. Where indicated, IPTG was added to the culture to induce the expression of yopE-dhfr. Transcript levels of yopB, lcrV, and rpoA were analyzed by RT-PCR. As a control for cDNA synthesis, reverse transcriptase (RT) was omitted from the reaction where indicated. (B) Y. enterocolitica W22703(pJS95) (black bars) and EC2(pJS95) [Δ(yscM1 yscM2)] (gray bars) cultures induced for type III secretion were pulse-labeled with 10 μCi/ml Pro-Mix, TCA precipitated, immunoprecipitated, and analyzed by SDS-PAGE and phosphorimaging. Relative amounts of protein synthesis were calculated by dividing the total amount of YopB and LcrV synthesized in strains expressing yopE-dhfr by the total amount of YopB and LcrV synthesized in strains not expressing yopE-dhfr. Values were normalized to the expression of RpoA and multiplied by 100%. Black and gray bars indicate the means of four independent experiments. Error bars indicate 1 standard deviation from the mean. Statistical significance was calculated using Student's t test. *, P < 0.01; **, P < 0.05.

FIG. 8.

YopE-DHFR cannot block temperature-sensitive growth of class II mutant Y. pestis. Y. pestis strain MEL2 (ΔlcrQ) was transformed with a vector control plasmid or with a plasmid expressing yopE-dhfr from the IPTG-inducible tac promoter (pJS95). Strains were streaked on HIA plates and incubated at 28 or 37°C with or without calcium and with or without IPTG, an inducer of the tac promoter driving yopE-dhfr expression.

FIG. 9.

YopE-DHFR cannot block type III injection of class II mutant Y. enterocolitica. (A) Strain EC2 [Δ(yscM1 yscM2)] carrying pJS93 was analyzed for type III injection into HeLa cells as described in the legend to Fig. 4. α, anti. (B) Data in panel A were analyzed to determine the percent amount of injected polypeptide. (C) Y. enterocolitica cytotoxicity for HeLa cells was measured by fluorescence microscopy and Texas red-phalloidin staining.

FIG. 10.

Overexpression of sycE does not rescue the inhibition of Yop synthesis in strains expressing yopE-dhfr. Strains of Y. enterocolitica expressing pJS95 (yopE-dhfr) were transformed with a high-copy-number plasmid that expresses sycE from the native sycE promoter. The resulting strains were induced for type III secretion by chelation of calcium. Cultures were centrifuged to separate medium in the supernatant (S) from bacterial sediment (P, pellet), and proteins were detected by immunoblotting. Where indicated, IPTG was added to the culture to induce the expression of yopE-dhfr. α, anti.

FIG. 11.

Removal of the YopE secretion signal abolished the YopE-DHFR blockade of the type III pathway. Y. enterocolitica W22703 expressing yopE-dhfr or yopE_Δ2-15-dhfr was induced for low-calcium type III secretion in the presence or the absence of IPTG. Cultures were centrifuged to separate medium in the supernatant (S) from bacterial sediment (P, pellet), and proteins were separated on a 15% SDS-PAGE gel and stained with Coomassie brilliant blue (A) or transferred to a PVDF membrane and immunoblotted with specific YopE, YopD, and NPT antibodies (B). α, anti.