Supplemental material

This file includes details for plasmid constructs,human cell lines, antibodies, and methods used only in supplemental figures and tables.

Plasmid constructs and mutagenesis

In plasmids for expression in E. coli or human cells, the lntA insert gene encodes residues G34 to K205 of the protein (without the signal peptide), whereas the complete open reading frame was cloned for Listeria-expression vectors. For the in vitro experiments, we used the following plasmids: pGEX-4T-1-BAHD1187-428, pGEX-4T-1-BAHD1239-361, pACYDuet-lntA, pGEX-4T-1-lntA, as well as site-specific mutants of these constructs. For transfection experiments we used the following plasmids: pEYFP-N1-BAHD1,pEYFP-N1-BAHD1∆cPRR[239-361], pcDNA3.1/V5-His-TOPO-lntA, and site-specific variants. Expression of lntA by Listeria was driven by the Pprot promoter on the pP1-lntA-V5 multicopy plasmid and its variants, as previously described (1). All lntA mutants were produced using the Quickchange II kit (Stratagene). Incorporation of the expected mutations into selected clones was confirmed by sequencing. A detailed list of plasmids used in this study is provided as Table S3.

Human cell lines

We used intestinal epithelial LoVo cells (CCL-229, ATCC), placental epithelial JEG-3 cells (HTB-36, ATCC), or 293FT derived from human embryonic kidney cells (R700-07, Invitrogen/Life technologies). All cell lines were grown following ATCC or Invitrogen/Life technologies recommendations, at 37°C in a humidified atmosphere containing 10% CO2.

Antibodies

The primary antibodies were as follows: Rabbit anti-GST antibody, HRP conjugated (A190-122P, Bethyl); mouse monoclonal anti-polyHistidine-peroxidase (A7058, Sigma); anti-V5 mouse monoclonal antibodies (R960-25, Invitrogen/Life technologies); rabbit anti-LntA (1); rabbit anti-LLO (2). For immunoblots, HRP-conjugated anti-GST and anti-polyHistidine at 1:2,000 dilution; anti-LntA and anti-LLO were used at a 1:10,000 and 1:50,000 dilution, respectively. Anti-V5 was used at 1:500 dilution in IF, 1:5,000 in immunoblot. Secondary antibodies were Cy3-conjugated goat anti-mouse IgGs (115-167-003, Jackson IR) for immunofluorescence (1:500 dilution), and HRP-conjugated goat anti-mouse or anti-rabbit IgGs (172-1011 or 172-1019, Bio-Rad) for immunoblots (1:10,000 dilution).

Purification of 6His-LntA mutants for biophysical characterization and gel filtration

6His-tagged LntAWT and LntAK180D/K181D were purified from BL21(DE3) cells grown at 25°C overnight after induction with 1mM IPTG. The cells were lysed in Buffer A with DNAse I and protease inhibitors (0.5g/ml of leupeptide, 0.7g/ml of aprotinin, 0.7g/ml of pepstatin and 1mM PMSF). Soluble fractions were loaded onto a 1 ml His-Trap column (GE Healthcare) equilibrated in 50mM NaPi pH 8.2, 0.2 M NaCl, 20mM imidazole, 5% glycerol and eluted with a linear gradient of the same buffer containing 500mM imidazole. The fractions containing LntA were pooled and concentrated before being loaded on a Superdex 200 HR30/10 column equilibrated in 20mM HEPES pH 6.5, 0.15 M NaCl, 1mM EDTA. Sample homogeneity was estimated by SDS-PAGE and Mass Spectrometry.

Thermal Shift Assay

Assays were conducted in an IQ5 96-well format real-time PCR instrument (Bio-Rad) in the presence of a Sypro Orange (Molecular Probes) probe. The total volume was 25μL with 2l of Sypro Orange probe, 50mM NaCl and 100mM of buffer (Na acetate pH4.6, Na citrate pH5.6, Na cacodylate pH6.5, HEPES pH7.5 or Tris-HCl pH8.5). Samples at 0.3 mg/ml, were heat-denatured from 20 to 100°C at a rate of 1°C per minute. At each step, excitation was performed at 470 nm, while emission of Sypro Orange fluorescence was monitored at 570 nm. Plotting of the fluorescence versus temperature curves, followed by the calculation of the first derivative at each point, allowed the identification of each inflection point; the minima were referred to as the melting temperatures (Tm).

1D-NMR analyses

Wild type LntA and LntAK180D/K181D, purified as described above, were dialyzed into 20mM Na acetate pH5.0, 50mM NaCl overnight at 4°C prior to analysis. A protein concentration of 2.2mg/ml was used for the three samples. Spectra were recorded at 25°C on a 600MHz spectrometer.

Analysis of Listeria extracts

Bacterial total extracts or culture supernatants were recovered from 1 ml of Listeria strains grown to a OD600 of 2.0 in BHI medium at 37°C, under microaerobic conditions. After centrifugation at 8,000× g, the bacterial pellet was resuspended in 50μl of Sample Buffer 2X (SB2X), and sonicated 5 times 30 seconds with a Bioruptor (Diagenode), leading to bacterial total extracts (TE). Supernatants (Sn) from centrifugation were filtered on 0.2μm filters, and then diluted with an equal volume of SB2X. Samples were analysed by SDS-PAGE and western blot.

Analysis of LntA protein sequences

A broad range sequence variability study of the lntA gene locus was undertaken on 188 L. monocytogenes isolates of diverse origins (human clinical cases, food, animal and environmental isolates)representative of all lineage (I, II, III) and serotype(1/2a-c 3b, 4a-e and 7). The complete list of strains used in this study and their characteristics is provided as Table S4.

The lntA locus was PCR amplified from genomic DNA from these isolates, using pairs of primers hybridizing 361 bp upstream and 695 bp downstream of the lntA start codon (lntAf: GCAACTGGAAAAGA; lntAr: GCAAGATCATGCTTGGCAG). The lntA gene was present in all L. monocytogenes isolates giving rise to PCR products the sequences of which were further determined. This revealed 19 distinct sequence variants (named A to S) of the LntA translated open reading frame. LntA sequence variants were aligned using Multalin(3).

The resulting 188 annotated DNA sequences of the lntA locus have been deposited in GenBank ( the accession numbers KJ027126 to KJ027313.

References

1.Lebreton A, Lakisic G, Job V, Fritsch L, Tham TN, Camejo A, Matteï P-J, Regnault B, Nahori M-A, Cabanes D, Gautreau A, Dessen A, Ait-Si-Ali S, Cossart P, Bierne H. 2011. A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response. Science 331:1319-1321.

2.Ribet D, Hamon M, Gouin E, Nahori MA, Impens F, Neyret-Kahn H, Gevaert K, Vandekerckhove J, Dejean A, Cossart P. 2010. Listeria monocytogenes impairs SUMOylation for efficient infection. Nature 464:1192-1195.

3.Corpet F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881-10890.

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