The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in normal rat kidney cells

Abstract

Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family.

Fig 1. Expression of ssej causes CPY-Inv to be mis-sorted in Saccharomyces cerevisiae.

(A) Fragment of the Salmonella chromosome inserted into the yeast expression vector causing CPY-Inv secretion. (B) Qualitative CPY-Inv secretion in yeast expressing individual Salmonella genes identified in (A). Negative control yeast (ctrl) contain just the cloning vector (pVT-100U) and positive control yeast lack the receptor VPS10 for CPY (ΔVPS10). (C) Quantitative CPY-Inv secretion in yeast expressing Salmonella genes. Controls as in (B). Data are from n = 3–9 (number of experiments for each condition in parentheses above each bar) and are mean ± S.D. *P<0.001 SseJ c.f. Ctrl (P>0.05 SseJ c.f. ΔVPS10). (D) Fluorescence visualisation of the yeast vacuole in wild type yeast (WT) transformed with vector (pVT-100U) alone or SseJ in pVT-100U (SseJ). Top panels DIC and bottom panels FM 4–64 fluorescence. Scale bar = 10 μm.

Fig 2. Re-distribution of late endocytic organelles in cells expressing SseJ.

(A) NRK cells expressing myc-SseJ were double labelled with anti-myc (a) and anti-lysosome glycoprotein 110 (Lgp110; b) followed by fluorescently labelled secondary antibodies. Panel c is the merged image of panels a and b, co-localisation is shown by yellow. (B) Control (Ctrl) NRK cells or NRK cells expressing myc-SseJ (myc-SseJ) were immuno-labelled for the mannose 6-phosphate receptor (MPR), Lgp110 and trans-Golgi network 38 (TGN38) followed by fluorescently-labelled secondary antibodies to visualise the late endosomes, lysosomes and trans-Golgi network respectively. (C) Aggregation of Lgp110 in NRK cells expressing SseJ for 24h (a). Quantification of cells showing aggregated lysosomes after induction of SseJ production with cadmium (Cd) (b). Expression of myc-SseJ protein -/+ Cd is shown by the western blot insert (b). (D) NRK cells were immunolabelled for microtubules (-alpha-tubulin; a,d), lysosomes (lgp120;b,e) and late endosomes (cation-independent mannose 6-phosphate receptor; c,f) in control cells (ctrl) or after cells had been treated with 10μM nocodazole for 1h. Scale bars represent 10μm.

Fig 3. Microtubules are disrupted in cells expressing SseJ.

(A) Control (Ctrl) NRK cells and cells expressing myc-SseJ (SseJ) or myc-SseJ-S151A (S151A) were fixed and the microtubules visualised using anti alpha-tubulin antibodies and fluorescently-labelled secondary antibodies. Bars = 10μm. (B) J774.2 mouse macrophages were either uninfected (Ctrl) or infected with WT or ΔsseJ Salmonella Typhimurium for 24h before fixing. The DNA (blue) was visualised using DAPI and the microtubules (red) were visualised as in A. Bars = 20μm. Quantification of the number of cells showing an organised microtubule network under each condition is shown (n = 1, scoring 100 cells per condition). (C) Cells as in A were fixed and de-tyrosinated alpha-tubulin (Glu-tubulin) visualised by immunolabelling using anti Glu-tubulin antibodies and fluorescently-labelled secondary antibodies. Bars = 10μm. (D) Cells as in A were lysed and lysates immunoblotted for acetylated-alpha-tubulin (Ac-tubulin) and alpha-tubulin. (E) myc-SseJ production was induced in NRK cells up to 24h. Lysates were generated and western blotted for myc-SseJ, acetylated-tubulin and Rho (pan specific). (F) NRK cells (Ctrl) and those expressing sseJ (SseJ) were transfected with a plasmid encoding EB3-tdTomato. EB3-tdTomato was visualised live, 24h later, on a spinning disc confocal microscope. Images represent a single time frame. Bars = 10μm.

Fig 4. SseJ binds GTPases RhoA and RhoC.

(A) Anti-myc antibody was covalently attached to sepharose and myc-SseJ was immunoprecipitated from control (Ctrl) NRK cells or NRK cells expressing myc-SseJ (myc-SseJ). Proteins bound to the beads were eluted and subjected to SDS-PAGE and the gel stained with coomassie (shown). SseJ is indicated by an arrowhead. A band at ≈21kDa specifically found in the SseJ immunoprecipitation was excised and sequenced by mass spectroscopy and identified both RhoA and RhoC. Peptides identified are shown by the insert with peptides common to both RhoA and RhoC shown in bold, peptides unique to RhoA shown in blue and peptides unique to RhoC shown by red. Only a single peptide was unique to RhoC (highlighted by an asterisk). (B) Experiments as shown in A, including cells expressing myc-SseJ(S151A), were repeated and western blotted for myc, RhoA and RhoC. Western blots show 1/10^th of the input before and after the immunoprecipitation and the total eluate from the immunoprecipitations. (C) The activity of RhoA was measured by ELISA, on extracts from control cells and cells expressing myc-SseJ or myc-SseJ (S151A) mutant. Data are means ± SD, n = 8.