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. 2004 Jul;72(7):4004-9.
doi: 10.1128/IAI.72.7.4004-4009.2004.

Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB

Gottfried Wilharm  1 , Verena Lehmann, Kristina Krauss, Beatrix Lehnert, Susanna Richter, Klaus Ruckdeschel, Jürgen Heesemann, Konrad Trülzsch
Affiliations

Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB

Gottfried Wilharm et al. Infect Immun. 2004 Jul.

Abstract

The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

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Figures

FIG. 1.
FIG. 1.
Motility of Y. enterocolitica WA-314 (wild type [WT]) and WA-314ΔmotAB on floating agar. (A) Yersiniae were grown on 0.3% agar (LB medium) with or without 30 μM CCCP and incubated overnight at 28°C. (B) Transcomplementation of WA-314ΔmotAB with plasmid pmotAB and influence on motility.
FIG. 2.
FIG. 2.
Y. enterocolitica type III secretion is sensitive to the protonophore CCCP. (A) Yersiniae were grown at 37°C for 2 h. Then, secretion of Yops was started by complexing Ca2+ with EGTA; simultaneously, CCCP was added (B) Alternatively, yersiniae were grown at 37°C for 2 h, preincubated with CCCP for 10 min, and subsequently stimulated with EGTA. Secretion was analyzed after 2 h of EGTA stimulation by TCA precipitation of the culture supernatant and subsequent SDS-PAGE (Coomassie staining).
FIG. 3.
FIG. 3.
Influence of CCCP on Y. enterocolitica growth (A), Yop secretion and expression (B), and ATP level (C). Six Yersinia cultures were grown in parallel for 2 h at 37°C in BHI. CCCP (10 μM) was added to three cultures, and DMSO (solvent for CCCP) was added to the other three cultures as a control. After 5 min, Yop secretion was induced by the addition of 5 mM EGTA and 10 mM MgCl2. Samples of supernatant and cell pellets were taken at 0, 30, and 60 min relative to the addition of CCCP or DMSO. The OD600 of cultures was monitored. (B) Culture samples with OD of >1 were diluted. Supernatant was precipitated with TCA, and equal amounts adjusted by OD (corresponding to approximately 1 ml of 1 OD600) were loaded onto an SDS gel and stained with Coomassie (upper panel). One fifth of corresponding cell pellets was loaded onto an SDS gel, electroblotted, and immunostained by using anti-YopE and anti-YopH antisera (lower panel). (C) For determination of intracellular ATP levels, 2 ml of bacterial cultures was centrifuged, and pellets were resuspended in 100 mM Tris-HCl (pH 7.75)-4 mM EDTA with adjustment of the OD600 of the cell suspension to 1.0. The cell suspensions (100 μl) were boiled for 2 min at 100°C. Samples were centrifuged, and 50 μl of each supernatant was transferred to a microtiter plate. Luciferase reagent (50 μl) was injected, and luminescence read by a luminometer at 20°C.
FIG. 3.
FIG. 3.
Influence of CCCP on Y. enterocolitica growth (A), Yop secretion and expression (B), and ATP level (C). Six Yersinia cultures were grown in parallel for 2 h at 37°C in BHI. CCCP (10 μM) was added to three cultures, and DMSO (solvent for CCCP) was added to the other three cultures as a control. After 5 min, Yop secretion was induced by the addition of 5 mM EGTA and 10 mM MgCl2. Samples of supernatant and cell pellets were taken at 0, 30, and 60 min relative to the addition of CCCP or DMSO. The OD600 of cultures was monitored. (B) Culture samples with OD of >1 were diluted. Supernatant was precipitated with TCA, and equal amounts adjusted by OD (corresponding to approximately 1 ml of 1 OD600) were loaded onto an SDS gel and stained with Coomassie (upper panel). One fifth of corresponding cell pellets was loaded onto an SDS gel, electroblotted, and immunostained by using anti-YopE and anti-YopH antisera (lower panel). (C) For determination of intracellular ATP levels, 2 ml of bacterial cultures was centrifuged, and pellets were resuspended in 100 mM Tris-HCl (pH 7.75)-4 mM EDTA with adjustment of the OD600 of the cell suspension to 1.0. The cell suspensions (100 μl) were boiled for 2 min at 100°C. Samples were centrifuged, and 50 μl of each supernatant was transferred to a microtiter plate. Luciferase reagent (50 μl) was injected, and luminescence read by a luminometer at 20°C.
FIG. 3.
FIG. 3.
Influence of CCCP on Y. enterocolitica growth (A), Yop secretion and expression (B), and ATP level (C). Six Yersinia cultures were grown in parallel for 2 h at 37°C in BHI. CCCP (10 μM) was added to three cultures, and DMSO (solvent for CCCP) was added to the other three cultures as a control. After 5 min, Yop secretion was induced by the addition of 5 mM EGTA and 10 mM MgCl2. Samples of supernatant and cell pellets were taken at 0, 30, and 60 min relative to the addition of CCCP or DMSO. The OD600 of cultures was monitored. (B) Culture samples with OD of >1 were diluted. Supernatant was precipitated with TCA, and equal amounts adjusted by OD (corresponding to approximately 1 ml of 1 OD600) were loaded onto an SDS gel and stained with Coomassie (upper panel). One fifth of corresponding cell pellets was loaded onto an SDS gel, electroblotted, and immunostained by using anti-YopE and anti-YopH antisera (lower panel). (C) For determination of intracellular ATP levels, 2 ml of bacterial cultures was centrifuged, and pellets were resuspended in 100 mM Tris-HCl (pH 7.75)-4 mM EDTA with adjustment of the OD600 of the cell suspension to 1.0. The cell suspensions (100 μl) were boiled for 2 min at 100°C. Samples were centrifuged, and 50 μl of each supernatant was transferred to a microtiter plate. Luciferase reagent (50 μl) was injected, and luminescence read by a luminometer at 20°C.
FIG. 4.
FIG. 4.
Immunofluorescence microscopy of yersinia (wild-type strain WA-314 left) and mutant WA-314ΔmotAB (right) flagella. Yersiniae were grown on minimal medium agar plates overnight at 28°C, gently harvested with PBS, and processed for immunofluorescent staining with anti-Y. enterocolitica flagellum serum and rhodamine-conjugated secondary anti-rabbit IgG antibodies.
FIG. 5.
FIG. 5.
Yop secretion of the Yersinia ΔmotAB mutant. Secretion of Yops by the wild-type strain and its motAB mutant was analyzed on a Coomassie-stained gel after secretion for 90 min.
FIG. 6.
FIG. 6.
Translocation of Yop effectors to HeLa cells is not affected by the deletion of motAB. The translocation-deficient yscV (lcrD) mutant served as a control. HeLa cells were infected at a multiplicity of infection of 50 for 2 h. Then, cells were detached by treatment with proteinase K, washed, and lysed by osmotic shock. Samples were centrifuged. S refers to supernatant after HeLa cell lysis and subsequent TCA precipitation thus corresponding to the fraction containing translocated Yops. P refers to the pellet fraction after HeLa cell lysis including adherent bacteria and cell debris. Fractions were loaded on an SDS gel, which was electroblotted. YopE and YopH were detected with appropriate antisera.
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