Research ArticleMICROBIOLOGY

Exploiting species specificity to understand the tropism of a human-specific toxin

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaax7515
DOI: 10.1126/sciadv.aax7515
  • Fig. 1 Binding of LukAB to CD11b I-domain of different species.

    (A) Phylogenetic tree based on amino acid sequences of CD11b I-domains from different species and percent amino acid identities to the human I-domain. (B) Coomassie staining of 1 μg of LukAB and I-domains. (C) Binding of LukAB to recombinant I-domains from the species examined in (A) as measured. Data are normalized to the maximum 450-nm absorbance of LukAB bound to the human I-domain. Data are represented as the average of three independent experiments ± SEM. Statistical significance was determined by two-way ANOVA (****P < 0.0001; **P < 0.01; *P < 0.05; ns, not significant) compared to murine I-domain. (D) Linear regression analysis of % binding (relative to human I-domain) at 1 μg/ml and percent amino acid identity to human I-domain.

  • Fig. 2 Positive selection on the CD11b I-domain resulting in changes in LukAB binding.

    (A) Sites undergoing episodic (MEME and PAML) and pervasive (FUBAR) positive selection in full-length CD11b are indicated by triangles. (B) Structure of the human I-domain [Protein Data Bank (PDB) ID: 1IDO]. Residues displaying signatures of positive selection in three independent algorithms [FUBAR (P > 0.9); PAML (M7 versus M8, P ≤ 0.05); MEME (P ≤ 0.05)] are shown as spheres and labeled. (C to E) Binding of LukAB to I-domain mutants (C) and I-domains from various primates (D) and rodents (E) as measured. Data are normalized to the maximum 450-nm absorbance of LukAB bound to the human I-domain. Data are represented as the average of three independent experiments ± SEM. Statistical significance was determined by two-way ANOVA (****P < 0.0001; ***P < 0.001; **P < 0.01). (F) Table of primates and rodents analyzed in (D) and (E) showing percent amino acid identity to the human I-domain and residues at sites 164, 222, and 294.

  • Fig. 3 I-domain chimeras reveal a critical region for LukAB binding.

    (A) Amino acid alignment of murine and human I-domains. Divergent amino acids are highlighted. Each color represents a different I-domain chimera. (B) Structure of human I-domain (PDB ID: 1IDO). Regions conserved with murine I-domain are shown in gray, and divergent regions are colored as in (A). (C to E) LukAB binding to human, mouse, HMH chimeric (murine residues in human I-domain backbone) (C to E), and MHM chimeric (human residues in murine I-domain backbone) (D and E) I-domains. HMH 7289–316 and MHM 7289–316 refer to HMH 7 and MHM 7 shown in (C and D), respectively (E). Data are normalized to the maximum 450-nm absorbance of LukAB bound to human I-domain. Data are represented as the average of three independent experiments ± SEM. Statistical significance was determined by two-way ANOVA (****P < 0.0001; ***P < 0.001; **P < 0.01) compared to human (C) or murine (D) I-domains. (F) Structure of human I-domain (PDB ID: 1IDO). Regions that are conserved with murine I-domain are shown in gray, and divergent regions are shown following the same color scheme as in (A) and (B). Residues 292 to 295 from chimera 7 are shown as spheres. (G) LukAB binding to HEK293T cells transfected with full-length CD11b and CD18. The percentage of LukAB bound to CD11b + cells was evaluated by flow cytometry. Data are represented as the average of three independent experiments ± SEM. Statistical significance was determined by two-way ANOVA (****P < 0.0001; ***P < 0.001; *P < 0.05) compared to murine CD11b. See also fig. S2.

  • Fig. 4 Characterization of the hCD11b mouse.

    (A) Schematic representation of murine Itgam locus and DNA template used to humanize exon 9. (B to D) iBMDMs from WT and hCD11b mice were stained for CD11b, F4/80, and MHC II and evaluated by flow cytometry (B). (C) WT and hCD11b iBMDMs incubated with GFP-producing S. aureus ± serum and phagocytosis measured as % iBMDMs GFP positive by flow cytometry. Data are represented as the average of three independent experiments performed in duplicate ± SEM. (D and E) WT and hCD11b iBMDMs (D) and peritoneal exudate cells (E) were incubated with biotinylated LukAB, and bound LukAB was quantified by flow cytometry. Data are represented as the average of three independent experiments performed in duplicate ± SEM. Statistical significance was determined by two-way ANOVA (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05). (F) Peritoneal exudate cells from WT and hCD11b mice were treated with LukAB, and propidium iodide (PI) incorporation was quantified by flow cytometry. Data are represented as the average of three independent experiments performed in duplicate ± SEM. Statistical significance was determined by two-way ANOVA (**P < 0.01 and *P < 0.05). (G) Peritoneal exudate cells treated with LukAB (12.5 μg/ml) were stained, and % PI-positive PMNs (CD11b+ and Ly6G+), macrophages (CD11b+ and F4/80+), monocytes (CD11b+, Ly6C+, and Ly6G), and DCs (CD11b+, CD11c+, and F4/80) were quantified by flow cytometry. Data are represented as the average of three independent experiments performed in duplicate ± SEM. Statistical significance was determined by one-way ANOVA (***P < 0.001).

  • Fig. 5 The hCD11b mouse is susceptible to MRSA infection.

    (A and B) WT and hCD11b mice were infected intravenously with ~3 × 106 CFU of WT USA300 strain LAC or an isogenic ΔlukAB LAC strain. Three days after infection, % of mice with detectable CFUs in the liver was determined (A). Statistical significance was determined by chi-square test (**P < 0.01). (B) CFUs in the livers were also quantified. Data are represented as the average of three independent experiments with 15 total mice per group. Statistical significance was determined by one-way ANOVA (**P < 0.01 and *P < 0.05). (C) WT and hCD11b mice were infected intravenously with 1 × 107 ΔlukAB::p LAC or ΔlukAB::plukAB LAC. One day after infection, CFUs in the livers were quantified. Data are represented as the average of two independent experiments with 10 total mice per group. Statistical significance was determined by one-way ANOVA (*P < 0.05). (D and E) hCD11b mice were infected intravenously with 1 × 108 CFU of WT LAC or an isogenic ΔlukAB LAC strain. (D) CFUs in the livers and kidneys 1 day after infection. (E) Tissue homogenates from (D) were also used to quantify LukAB. Data are represented as the average of three independent experiments with 15 total mice per group. Statistical significance was determined by one-way ANOVA (**P < 0.01). See also fig. S4. (F) WT mice were infected with S. aureus intravenously. One day after infection, the % Ly6G+ CD11b+ PMNs out of the total CD45% leukocytes were quantified. Data are represented as the average of two independent experiments with six total mice per group. Statistical significance was determined by one-way ANOVA (***P < 0.001).

  • Table 1 SPR data of LukAB binding to recombinant I-domains.

    Protein + LukABKD
    Human39.29 ± 8.12 nM
    Mouse246.6 ± 121.9 μM
    HMH 778.10 ± 21.9 μM
    MHM 769.60 ± 19.51 nM
    MHM 7292–295173.70 ± 18.9 nM

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaax7515/DC1

    Fig. S1. Purification of CD11b I-domains.

    Fig. S2. Purification and transfection of CD11b I-domain chimeras.

    Fig. S3. Characterization of the hCD11b mouse.

    Fig. S4. LukAB exhibits liver-specific tropism in vivo.

    Table S1. Signatures of positive selection on primate ITGAM.

    Table S2. Signatures of positive selection on rodent ITGAM.

    Table S3. Primate and rodent sequences analyzed for selection on ITGAM.

    Table S4. S. aureus strains used in this study.

    Table S5. Plasmids used in this study.

    Table S6. Oligonucleotides and gene sequences used in this study.

    References (3840)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Purification of CD11b I-domains.
    • Fig. S2. Purification and transfection of CD11b I-domain chimeras.
    • Fig. S3. Characterization of the hCD11b mouse.
    • Fig. S4. LukAB exhibits liver-specific tropism in vivo.
    • Table S1. Signatures of positive selection on primate ITGAM.
    • Table S2. Signatures of positive selection on rodent ITGAM.
    • Table S3. Primate and rodent sequences analyzed for selection on ITGAM.
    • Table S4. S. aureus strains used in this study.
    • Table S5. Plasmids used in this study.
    • Table S6. Oligonucleotides and gene sequences used in this study.
    • References (3840)

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