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. 2023 Jan 25;26(2):106042. doi: 10.1016/j.isci.2023.106042

Figure 7.

Figure 7

Internalization of Mab by macrophages requires the AhpC adhesin

(A) Schematic illustration of the Mab ahpC conditional knock-out strategy (see also Table S1). Mab was transformed with the regulatory, ha-tagged ahpCi plasmid (pMV306-ahpCi) to generate a merodiploid bacterial strain. The pMV306-ahpCi vector was integrated into the attB site by single homologous recombination. The pMV306-ahpCi contains the integrase gene (int), the hygromycin resistance cassette (HygR), the tetR38 repressor under the control of the Ptb38 promoter, and the C-terminal ha-tagged ahpC gene under the control of the inducible tetO-4C5G promoter. The single merodiploid clone was then transformed with pUX1-katG-ahpC to remove the endogenous ahpC gene (ahpCe) by double homologous recombination. The ahpC gene is sandwiched between the ahpD and MAB_4409. The DNA sequences of the left and right arms of ahpC were amplified by PCR and subcloned into pUX1-katG. The resulting suicide plasmid was used to transform the strain expressing an additional HA-tagged copy of ahpC gene. Dotted lines represent the size (indicated above each line) of the expected PCR products in Mab WT, and ΔahpCe/ahpCi-ha. Black arrows represent the primers used for PCR analysis.

(B) Tetracycline-mediated regulation of the ahpC gene using the tet-OFF configuration in Mab. The tetR38 gene encoding the T38 repressor is a reverse TetR that recognizes tetO-4C5G. Addition of ATc results in binding of T38 to ATc and its recruitment to tetO-4C5G represses the transcription of the HA-tagged ahpCi gene. T38, Tet repressor; ATc, anhydrotetracycline; tetO, Tet operator.

(C) PCR analysis confirming the deletion of ahpC in ΔahpCe/ahpCi-ha. Genomic DNA from Mab was used to amplify the intact ahpC locus. Amplicon was subjected to sequencing to confirm the proper deletion of ahpC.

(D) Time-dependent depletion of the inducible AhpCi-HA upon addition of ATc to the medium is shown after 24 h, 48 h, and 72 h in ΔahpCe/ahpCi-ha (upper panel). KasA was used as a loading control (lower panel).

(E) Parental Mab and ΔahpCe/ahpCi-ha strains growth curves in 7H9 media supplemented or not with ATc at 37°C. The data are from one of two independent experiments.

(F) Bacteria were grown to exponential phase and 1.5 μL of 10-fold serial dilutions were spotted onto LB agar medium supplemented or not with 1 μg/ml ATc. Pictures were taken after 5 days of incubation at 37°C. Arrows indicate the difference of growth in presence or absence of ATc.

(G) Comparison of the invasion capacity of the parental and ΔahpCe/ahpCi-ha strains pre-exposed to 1 μg/mL ATc for 72 h and subsequently used to infect THP-1 cells. CFUs were determined at 3 hpi as reported earlier. The results represent the mean values ±SD of three independent experiments (each conducted in triplicate) (n = 9). One-tailed Tukey’s test: ns, non-significant > 0.05, ∗∗∗p < 0.001.