A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells

Abstract

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

Fig. 1.

The Beps mediate VirB/VirD4-dependent invasion, antiapoptotic protection, proinflammatory activation, and control of proliferation of HEC. (A) Structure of the virB/virD4/bep locus encoding the VirB components (VirB2-VirB11), the T4S coupling protein (VirD4), and seven putative effector proteins (BepA-G). (B) Domain structure of BepA-G. Yellow boxes represent tyrosine-containing sequence repeats resembling tyrosine-phosphorylation motifs (indicated by Y). (C) VirB4/VirD4/Bep proteins are required for mediating characteristic actin rearrangements, which result in uptake of Bh aggregates by means of invasomes. HUVEC infected with the indicated Bh strains were stained for F-actin. (Scale bar, 10 μm.) (D) VirB4/VirD4/Bep proteins are required for antiapoptosis. Caspase-3/7 activity of HUVEC was measured after infection with the indicated Bh strains for 24 h, followed by induction of apoptosis by actinomycin D for the indicated times. (E) VirB/VirD4/Bep proteins are required for NF-κB-dependent proinflammatory activation. HUVEC were infected for the indicated time with the indicated Bh strains, followed by quantification of IL-8 in the culture medium. (F) VirB4/VirD4/Bep proteins are required for controlling Bh-stimulated HUVEC proliferation. HUVEC infected with the indicated Bh strains were counted at the indicated time points, and proliferation indices were calculated. (D-F) Triplicate samples ± standard deviation.

Fig. 2.

BepD becomes tyrosine-phosphorylated after VirB4-dependent translocation into HEC. (A) VirB4-dependent translocation of BepD into HEC results in tyrosine phosphorylation and a coincident reduction in electrophoretic mobility. Total protein extracts of Ea.hy926 cells uninfected (lane 1) or infected with ΔvirB4 (lane 2), wild type (lane 3), ΔvirB4/pPG104 (lane 5), or wild type/pPG104 were prepared. FLAG-BepD encoded by pPG104 was immunoprecipitated with anti-FLAG antibodies, separated by SDS/PAGE, and immunoblotted with anti-FLAG (Left) or anti-phosphotyrosine antibodies (Right). (B and C) Immunocytochemical detection of FLAG-BepD after VirB/VirD4-mediated translocation into HEC. Ea.hy926 cells were infected with wild-type (B) or the ΔvirB4 mutant (C), each harboring pPG104. Specimens were immunocytochemically stained for the FLAG epitope (green), F-actin (red), and bacteria (blue). (Scale bar, 10 μm.)

Fig. 3.

The C-terminal translocation signal of Beps mediates VirB/VirD4-dependent protein transfer into HEC. Protein transfer was determined by CRAFT. The Cre-tester cell line Ea.hy926/pRS56-c#B1 was infected with the indicated Bh strains expressing different NLS-Cre fusion proteins (plasmid names are indicated in blue in A-E or black in F). The region of a given Bep fused to the C terminus of NLS-Cre is specified by the respective first and last amino acids (except for pRS96, which expresses only NLS-Cre). Percentages of GFP-positive cells as determined by FACS analysis are indicated in red. (A) NLS-Cre fused to the C-terminal 183 aa of BepD translocates efficiently into HEC in a VirB/VirD4-dependent manner. Dot blots of forward scatter (FSC) and GFP fluorescence (FL-1) are shown for the indicated Bh strains. (B) Relative translocation efficiency mediated by the BID domain of BepB, BepC, and BepF. (C) The signal for VirB/VirD4-dependent translocation into HEC is bipartite, composed of the BID domain and an adjacent unconserved C-terminal tail. (D) Creation of an efficient bipartite translocation signal by fusing a BID domain of BepF and the C-terminal tail of BepD. (E) The C terminus of the relaxase TraA of At plasmid pATC58 contains a BID domain and mediates efficient protein transfer from Bh into HEC. (F) Steady-state NLS-Cre fusion protein levels in Bh grown on isopropyl β-d-thiogalactoside-containing medium.

Fig. 4.

The coupling proteins (VirD4/TraG) of T4S systems containing a BID domain in their protein substrate(s) form a distinct phylogenetic cluster. This cluster is formed by Bartonella VirB/VirD4 systems and α-proteobacterial conjugative DNA transfer systems (cluster A) and does not contain agrobacterial T-DNA transfer systems (cluster B). VirD4/TraG protein sequences were extracted from the Uniprot database and then aligned and diagrammed as an unrooted neighbor-joining radial tree. T4S systems containing a BID domain in one of their substrate(s) are marked in bold (compare with Table 5). The following sequences (with corresponding accession numbers) are included. At: plasmid pAT (Q8UKJ4), pTiC58 (Q44346 and P18594), pTiA6NC (Q44360 and P09817), pRi1724 (Q9F5E3 and Q9F585), pTi15955 (Q8VLK3), and pTiAB2/73 (Q8VT85); Agrobacterium rhizogenes: pRiA4b (Q93UY7 and P13464); Bh (Q6G2A8); Bartonella quintana (Q6FYV9); Bartonella tribocorum (Q8GJ55); Escherichia coli: pKM101 (Q46706) and R388 (Q04230); Hp (Q75XB9); Lp (Q9RLR2); Oligotropha carboxydovorans (Q6LB53); Rhizobium etli: p42d (Q8KL68); Rhizobium meliloti: pSymA (Q92ZI3); and Rhizobium spp.: pNGR234a (P55421).