Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

Abstract

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

Keywords: Salmonella; SseK1; TBCB; arginine N-glycosyltransferase; tubulin; type III secretion.

Figure 1

Human tubulin-binding cofactor B (TBCB) interacts with SseK1 in the yeast two-hybrid system. Strain L40 of S. cerevisiae was cotransformed with derivatives of plasmids pLEX10, to generate fusions with the DNA-binding domain of LexA, and pGAD1318, to generate fusions with the activation domain of Gal4, as indicated. The interaction between the two hybrid proteins is shown by the growth in the absence of histidine (-His), and the detection of blue color in the presence of X-Gal after a β-galactosidase filter assay. Empty vectors were used as negative controls.

Figure 2

TBCB interacts with SseK1 in bacteria and host cells. (A) Bacterial lysates from strain SV7071 (14028 sseK1::3xFLAG) expressing glutathione S-transferase (GST) or GST-TBCB were prepared in NP40 buffer. Then, purified GST fusion proteins were blotted on nitrocellulose membranes, stained with Ponceau S red (left panel), and developed with monoclonal anti-FLAG (right panel). Aliquots of the original lysates were also included. The molecular weights of the marker bands (M) are indicated. (B) HeLa cells were transiently transfected with a derivative of pBABEpuro expressing SseK1-3xFLAG, a derivative of pCS2 expressing 3xHA-TBCB or cotransfected with the same plasmid and a derivative of pBABEpuro expressing SseK1-3xFLAG. NP40 lysates from 6 × 10⁶ transfected cells were subjected to immunoprecipitation (IP) with anti-FLAG monoclonal antibodies and, after washing, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and developed with monoclonal anti-HA. Total lysates from 5 × 10⁵ transfected cells are included in the blot. M, molecular weight marker. Molecular weights in kDa are indicated on the left.

Figure 3

SseK1 glycosylates TBCB in vitro. Assay for SseK1 GlcNAc modification of TBCB using recombinant GST fusion proteins in the indicated combinations and 1 mM UDP-GlcNAc. SseK1_AAA: SseK1(D223A/D225A). TBCB_N: N-terminal fragment of TBCB (amino acids 1–125). TBCB_C: C-terminal fragment of TBCB (amino acids 126–244). A representative immunoblot is shown incubated with anti-Arg-GlcNAc (upper panel). The same blot reincubated with anti-GST was used as the loading control (lower panel). Molecular weights in kDa are indicated on the left.

Figure 4

SseK1 glycosylates TBCB in vivo. (A) NP40 lysates from HEK293T cells transiently transfected with plasmids expressing GFP-SseK1 and/or 3xHA-TBCB, as indicated, were analyzed by immunoblot using anti-Arg-GlcNAc antibodies. (B) Lysates from HEK293T transiently transfected with a plasmid expressing GFP-SseK1 or with this plasmid and another plasmid expressing 3xHA-TBCB were subjected to immunoprecipitation (IP) with an anti-HA antibody. Precipitates were analyzed by western blot (W) with an anti-Arg-Glc-NAc antibody. Molecular weights in kDa are indicated on the left. The arrow indicates the band corresponding to TBCB.

Figure 5

SseK3 interacts with TBCB in the yeast two-hybrid system. Strain L40 of S. cerevisiae was cotransformed with derivatives of plasmids pLEX10, to generate fusions of SseK1, SseK2, or SseK3 with the DNA-binding domain of LexA, and pGAD1318, to generate a fusion of TBCB with the activation domain of Gal4, as indicated. The interaction between the two hybrid proteins is shown by the growth in the absence of histidine (-His), and detection of blue color in the presence of X-Gal after a β-galactosidase filter assay.

Figure 6

SseK2 and SseK3 are unable to glycosylate TBCB in vitro. An assay for GlcNAc modification of TBCB in the presence of SseK2 or SseK3 using recombinant GST fusion proteins in the indicated combinations and 1 mM UDP-GlcNAc. SseK3_AAA: SseK3(D226A/D228A). TBCB_N: N-terminal fragment of TBCB (amino acids 1–125). TBCB_C: C-terminal fragment of TBCB (amino acids 126–244). An immunoblot incubated with anti-Arg-GlcNAc is shown (upper panel). The same blot was reincubated with anti-GST (lower panel). Molecular weights in kDa are indicated on the left.

Figure 7

SseK1 increases the acetylation level of tubulin in HEK293T cells. (A) Representative fluorescence microscopy images showing GFP (Control) or GFP fusion proteins (GFP-SseK1 or GFP-SseK1_AAA,) in green, and acetyl-α-tubulin in red. HEK293T cells were transfected with derivatives of plasmid pEGFPC1, and 48 h after transfection cells were fixed with 4% paraformaldehyde and stained with anti-acetyl-α-tubulin rabbit monoclonal antibody and Alexa Fluor Plus 594-conjugated anti-rabbit secondary antibody. (B) Quantification of acetyl-α-tubulin from immunofluorescence images of cells transfected with GFP (Control) or GFP fusion proteins (GFP-SseK1, GFP-SseK1_AAA). The mean and standard deviation from 100 cells are represented for each condition. RFU, relative fluorescence units. ***, P < 0.001; ****, P < 0.0001, for the indicated comparisons using a Kolmogorov–Smirnov test.