PMC

Structural comparison of LntA^WT and LntA^K180D/K181D. (A) Comparison of charged patches on the surfaces of LntA^WT (left) and LntA^K180D/K181D (middle). On the right, a ribbon overlay shows LntA^WT in light teal and LntA^K180D/K181D in light pink. The mutated residues are indicated as sticks. (B) Arrows indicate the direction of an elongated groove in the structures, which ends with the basic patch affected by the mutations.

Positive charges in patch 3 of LntA are critical for interaction with BAHD1. (A) 6His-tagged LntA^WT and LntA mutants coexpressed with GST-BAHD1^187–428 in E. coli were used for GST pulldown assays. 6His-LntA^WT alone and 6His-LntA^WT coexpressed with GST-BAHD1^239–361 were used as negative controls. Proteins were detected by immunoblotting against the tags. (B) GST-tagged LntA^WT, LntA mutants, or InlC bound to glutathione-Sepharose was incubated with chromatin extracts containing full-length BAHD1-V5 or CFP-V5 as a negative control. V5-tagged fusions were detected by immunoblotting. GST-fused baits were detected by colloidal Coomassie blue staining.

The surface of LntA displays two major basic patches and one acidic patch. (A) LntA folds into a compact helical structure that is stabilized by a ladder of hydrophobic residues, indicated as yellow sticks. The thermal-motion color scale (B-factor values) indicates that the body formed by H1, H2, and H3 is the less mobile region and that the H4-H5 elbow displays more flexibility. Nter, N terminus. (B) The three main charged patches targeted by mutagenesis are shown (green circles) on the electrostatic surface views of LntA. Basic regions are in blue and acidic regions in red. Patch-forming residues are shown as sticks.

The K180/K181 dilysine motif in LntA and the proline-rich region in BAHD1 are required for the colocalization of the two proteins in human cells. 293FT cells were cotransfected for 48 h with WT or mutant versions of pcDNA3-lntA-V5 and pEYFP-N1-BAHD1. For lntA-V5, alleles resulted in WT, K180D/K181D, or K180R/K181R proteins. For BAHD1-YFP, alleles resulted in WT or ∆cPRR (i.e., missing residues 239 to 361) proteins. LntA-V5 and BAHD1-YFP were detected by immunofluorescence confocal microscopy. Arrows indicate the position of an Xi. A magnification of these regions with enhanced contrasts is shown on the left. Bars, 10 µm for the cell view and 1 µm for magnified Xi regions.

Stimulation of ISG expression by LntA during infection is impaired by mutation of the K180/K181 dilysine motif. Innate immune gene expression was quantified by RT-qPCR in noninfected LoVo cells (NI) or cells infected for 18 h with L. monocytogenes lntA^∅, lntA^WT, lntA^K180R/K181R, or lntA^K180D/K181D (left). Induction of the expression of interleukin 8 (IL-8), IFI6, and IFITM1 by L. monocytogenes lntA^∅ and L. monocytogenes lntA^WT, compared to uninfected cells (right). LntA-dependent induction of ISGs, monitored by normalization to cells infected with L. monocytogenes lntA^∅. Error bars indicate standard deviations. The reference gene for normalization was YWHAZ; GAPDH was a control invariant gene. Statistical analysis was performed using analysis of variance (ANOVA) followed by the Tukey-Kramer posttest. ns, nonsignificant; ***, P < 0.001.

LntA interacts in vitro with BAHD1^187–428. (A) Primary structure of BAHD1. GST-BAHD1^187–428 and GST-BAHD1^239–361 fusions were tested for interaction with LntA. cPRR, central proline-rich region; NLS, nuclear localization signal; BAH, bromo-adjacent homology domain. (B) Gel filtration experiment. Purified LntA, GST-BAHD1^187–428, or GST-BAHD1^239–361, incubated alone or coincubated for 3 h at 4°C, were injected on a Superdex 200 column. Elution fractions (lanes 1 to 9) were analyzed by SDS-PAGE followed by Coomassie blue staining. Elution fractions of single proteins are shown in the first three lines; those of coincubated proteins are highlighted in the gels labeled a and b. *, coelution of LntA and GST-BAHD1^187–428. (C) 6His-LntA was coexpressed in E. coli with GST-BAHD1^187–428 or GST-BAHD1^239–361. Input and eluted fractions from pulldown assays on His-Trap or GST-Trap columns were analyzed by immunoblotting against the GST or the 6His tag.