Apolipoprotein A-I exerts bactericidal activity against Yersinia enterocolitica serotype O:3

Abstract

Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), is well recognized for its antiatherogenic, antioxidant, and antiinflammatory properties. Here, we report a novel role for apoA-I as a host defense molecule that contributes to the complement-mediated killing of an important gastrointestinal pathogen, Gram-negative bacterium Yersinia enterocolitica. We specifically show that the C-terminal domain of apoA-I is the effector site providing the bactericidal activity. Although the presence of the lipopolysaccharide O-antigen on the bacterial surface is absolutely required for apoA-I to kill the bacteria, apoA-I does not interact with the bacteria directly. To the contrary, exposure of the bacteria by serum proteins triggers apoA-I deposition on the bacterial surface. As our data show that both purified lipid-free and HDL-associated apoA-I displays anti-bacterial potential, apoA-I mimetic peptides may be a promising therapeutic agent for the treatment of certain Gram-negative infections.

FIGURE 1.

Blocking of apoA-I in serum with specific antibodies and survival of Y. enterocolitica bacteria in 10% NHS. Wild-type Y. enterocolitica strain (YeO3; A) as well as its mutants lacking YadA (YeO3-O28; B), LPS_YeO3 O-antigen (YeO3-R2; C), or both (YeO3-O28-R1; D) were incubated in NHS alone or supplemented with (i) rabbit antiserum against human apoA-I, (ii) IgG fraction of rabbit apoA-I antiserum (R283), (iii) mAb 8G8 against apoA-I, or (iv) control antibody represented by preimmune rabbit IgGs. Data represent means ± S.D. (error bars) from three separate experiments, each done in duplicate. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ns, not significant.

FIGURE 2.

Interaction between apoA-I and LPS_YeO3. A, gel filtration analysis of the apoA-I-LPS_YeO3 complex. Human apoA-I (1.5 mg/ml), HDL₂ (1.5 mg/ml, as protein), or HDL₃ (1.5 mg/ml, as protein) were preincubated with smooth LPS (50 μg/ml) and applied into a Superose 6 column (HR 10/30; Pharmacia Biotech). The control contained the corresponding amount of LPS incubated in the presence of PBS. The collected fractions were analyzed for the presence of apoA-I and LPS_YeO3 using immuno-dot blotting with mAb TomA6 and R297 Abs. Arrows denote the elution positions of marker proteins: thyroglobulin, 670 kDa; bovine γ-globulin, 158 kDa; chicken ovalbumin, 44 kDa; equine myoglobin, 17 kDa; vitamin B12, 1.4 kDa. B, immunoprecipitation of the apoA-I-LPS_YeO3 complex from the human serum. Protein G-Sepharose-coupled rabbit IgGs against apoA-I (R322) were used to immunoprecipitate apoA-I-complexes formed in 10% NHS that had been preincubated with LPS_YeO3 (10 μg/ml) or PBS. The bound material was eluted (0.1 m glycine, pH 2.5) and loaded onto the Superose 12 column. Fractions were analyzed for the presence of apoA-I and LPS_YeO3 using immuno-dot blotting. C, immunoblotting analysis of the apoA-I binding to Y. enterocolitica strains. YeO3-O28 and YeO3-O28R strains were incubated with of 50% NHS for 5, 10, 15, or 20 min at 37 °C. Bacteria incubated with PBS for 20 min at 37 °C served as control. After extensive washings, bound apoA-I was detected by immunoblotting with anti-apoA-I antibodies (R315). D, immunofluorescence analysis of apoA-I binding to Y. enterocolitica. YeO3-O28 and YeO3-O28R strains incubated with 10% HIS, apoA-I (0.15 mg/ml), HDL₃ (0.15 mg/ml) for 40 min at 37 °C. Following three washes, the bacteria were incubated with 7.5 μg of R315 rabbit IgG against apoA-I. Bound antibodies were detected with Alexa Fluor 488 donkey anti-rabbit antibody.

FIGURE 3.

ApoA-I targets the LPS O-antigen of Y. enterocolitica. NHS was used at a final concentration of 10%. A, survival of Y. enterocolitica indicator bacteria in the LPS_YeO3-treated NHS. The bacteria were incubated in (i) NHS alone, (ii) NHS preincubated with 10 μg of 37 °C smooth or rough LPS_YeO3, or (iii) HIS. B, LPS_YeO3 treatment of NHS blocking apoA-I bactericidal activity. Y. enterocolitica indicator bacteria were incubated in (i) NHS alone, (ii) NHS pretreated with 5 μg of 10 °C smooth LPS_YeO3, (iii) 10 °C LPS_YeO3-treated NHS supplemented with a physiological amount of apoA-I (0.15 mg/ml), or (iv) HIS. C (right), abundance of the O-antigen in the LPSs isolated from the Y. enterocolitica outer core mutants (YeO3-trs24, YeO3-trs22, and YeO3-trs11) grown at 22 °C or 37 °C. The O-antigen content in the isolated LPSs was determined by the immuno-dot blotting using mAb TomA6 against the O-antigen. Quantitative analyses were performed using ImageJ software. C (left), apoA-I blocking potential of LPSs varying in the O-antigen content. The Y. enterocolitica indicator bacteria were incubated in (i) NHS alone, (ii) NHS pretreated with 5 μg of LPSs isolated from the Y. enterocolitica outer core mutants (YeO3-trs24, YeO3-trs22, and YeO3-trs11) grown at 22 °C or 37 °C, (iii) NHS pretreated with one of the above-mentioned LPSs supplemented with physiological amounts of apoA-I, or (iv) HIS. D, serotype O:3O-antigen flagging the Y. enterocolitica serotype O:8 bacteria for the apoA-I-mediated bactericidal attack. Y. enterocolitica serotype O:8 strain (8081-c-R2/pAY100:Tet) that expresses serotype O:3 O-antigen was incubated in (i) NHS, (ii) 10 °C LPS_YeO3-treated NHS, (iii) 10 °C LPS_YeO3-treated NHS supplemented with physiological amounts of exogenous apoA-I, or (iv) HIS. E, effects of HDL, LDL, and VLDL on Y. enterocolitica survival in human serum. The Y. enterocolitica indicator bacteria were incubated in (i) NHS, (ii) NHS pretreated with 0.5, 2, or 5 μg of 10 °C LPS_YeO3, (iii) 10 °C LPS_YeO3-treated NHS to which 0.15 mg/ml wild-type apoA-I, HDL₂, or HDL₃ was added, (iv) 10 °C LPS_YeO3-treated NHS to which 0.11 mg/ml LDL or VLDL was added, or (v) HIS. Data represent means ± S.D. (error bars) from two separate experiments, each done in duplicate. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ns, not significant.

FIGURE 4.

Mapping the apoA-I domain necessary for the apoA-I-mediated bactericidal activity in 10% NHS. A (right), secondary structure of the lipid-free apoA-I-based computer modeling (43). Epitopes of mAbs specific for apoA-I (4H1, 3G10, M9, 8A4, 7C5, or 4.1) are indicated. A (left), mapping the apoA-I domain responsible for the bactericidal action in human serum using anti-apoA-I mAbs. The Y. enterocolitica indicator bacteria were incubated with (i) NHS, (ii) NHS to which 12 μg of mAb against apoA-I (4H1, 3G10, M9, 8A4, 7C5, or 4.1) was added, or (iii) HIS. B, bactericidal potential of the apoA-I mutants. The Y. enterocolitica indicator bacteria were incubated with (i) NHS, (ii) NHS pretreated with 5 μg of 10 °C LPS_YeO3, (iii) 10 °C LPS_YeO3-treated NHS to which 0.15 mg/ml wild-type or mutant apoA-I (Δ220–243, Δ185–243, Δ1–59, Δ185–243, L218A/L219A/V221A/L222A, and E223A/K226A) was added, or (iv) HIS. Data represent means ± S.D. (error bars) from three (A) or two (B) separate experiments, each done in duplicate. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ns, not significant.

FIGURE 5.

Complement system and the bactericidal action of apoA-I in 10% serum. A (left), efficacy of EDTA to block the complement activation. The Y. enterocolitica indicator bacteria were incubated with (i) NHS, (ii) NHS treated with 0.5 mm EDTA, (iii) EDTA-treated NHS supplemented with 2 mm CaCl₂ and 0.5 mm MgCl₂, or (iv) HIS. A (right), EDTA-blocked complement activation and apoA-I activity. The Y. enterocolitica indicator bacteria were incubated with (i) NHS, (ii) NHS pretreated with 5 μg of 10 °C LPS_YeO3, (ii) 10 °C LPS_YeO3-treated NHS supplemented with physiological amounts of apoA-I, (iii) 10 °C LPS_YeO3-treated NHS to which 0.5 mm EDTA was added, (iv) apoA-I-supplemented, 10 °C LPS_YeO3-treated NHS containing only EDTA, or EDTA supplemented with 2 mm CaCl₂ and 0.5 mm MgCl₂, (v) HIS. B (left), efficacy of the anti-C5 antibody to block the complement activity. The Y. enterocolitica indicator strain was incubated with (i) NHS, (ii) NHS supplemented with the mAb against C5 (5 μg/ml) or its isotype-matched control, anti-HIV1 gp120 antibody (5 μg/ml), or (iii) HIS. B (right) and C, apoA-I activity in anti-C5 antibody-treated serum. The Y. enterocolitica indicator strain was incubated with (i) NHS, (ii) NHS pretreated with 5 μg of 10 °C LPS_YeO3, (ii) 10 °C LPS_YeO3-treated NHS supplemented with physiological amounts of apoA-I, (iii) 10 °C LPS_YeO3-treated NHS to which anti-C5 (5 μg/ml, B; 1.5, 2.5, or 5 μg/ml, C) or anti-gp120 was added (5 μg/ml, B; 1.5, 2.5, or 5 μg/ml, C), (iv) apoA-I-supplemented, 10 °C LPS_YeO3-treated, NHS containing anti-C5 or anti-gp120 antibodies, (v) HIS. Data represent means ± S.D. (error bars) from two separate experiments, each done in duplicate. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ns, not significant.