Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

Protokol

1. Analysis of Vitronectin, Vn as a Bacterial Surface Protein-Ligand

1. Detection of Vn-binding at the bacterial surface using flow cytometry
   NOTE: In flow cytometry, we used side scatter and forward scatter to gate positive events. To examine the interactions with Vn, Haemophilus influenzae type f, Hif clinical isolates (n=10) were selected together with E. coli BL21 (DE3) as a negative control (Figure 1A).
   1. Culture Hif clinical isolates in brain-heart infusion (BHI) medium supplemented with 10 µg/mL nicotinamide adenine dinucleotide (NAD) and hemin at 37 °C with shaking at 200 rpm. Use Luria-Bertani medium to culture E. coli. Harvest Hif at mid-log phase (Optical density, OD₆₀₀ = 0.3), and resuspend the bacteria in phosphate-buffered saline (PBS), pH 7.2, containing 1% (w/v) bovine serum albumin (BSA) (blocking buffer; PBS-BSA). Adjust the suspension to 10⁹ colony-forming units, CFU/mL.
   2. Transfer the aliquots containing 5 x 10⁶ CFU to 5 mL polystyrene round-bottom tubes (12 x 75 mm²) and add 1 mL of blocking buffer. Centrifuge the suspension at 3,500 × g at room temperature (RT) for 5 min to pellet the bacteria, then carefully aspirate to remove the supernatant without disturbing the pellet.
   3. Resuspend the bacterial pellets with 50 μL of blocking buffer containing 250 nM Vn and incubate the samples for 1 h at RT without shaking. After incubation, pellet the bacteria by centrifugation at 3,500 x g for 5 min, then wash the pellets three times using PBS and similar centrifugation steps.
   4. To the bacterial pellet, add 50 μL of primary sheep anti-human Vn polyclonal antibodies (pAbs) at 1:100 dilution in PBS-BSA. Incubate the suspension for 1 h at RT, then wash the bacteria three times with PBS to remove unbound antibodies (as described in step 1.1.3).
   5. Next, add 50 μL of PBS-BSA containing fluorescein isothiocyanate (FITC)-conjugated donkey anti-sheep pAbs (1:100 dilution) and incubate at RT for 1 h in the dark.
   6. Prepare a blank control by incubating bacteria with blocking buffer only and pAbs without Vn.
   7. Wash bacteria three times with 1 mL of blocking buffer and pellet the suspension by centrifugation, as described in section 1.1.2. Finally, resuspend the bacterial pellet with 300 µL of PBS and analyze it by flow cytometry.

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Sonuçlar

Figure 1: Haemophilus influenzae serotype f binds Vn via surface-expressed PH. (A) Flow cytometry results showing binding of Vn to cells of Hif clinical isolates. Each clinical isolate (5 x 10⁶ CFU) was incubated with 250 nM human Vn. The bound ligand was detected using sheep anti-Vn pAbs and FITC-conjugated donkey anti-sheep secondary antibodies. Escherichia coli BL21 (DE3) was included as a negative control. Data are presented as the mean fluorescence intensity from triplicate analyses in three separate experiments, and error bars represent SD. (B) Flow cytometry histograms demonstrating binding of Vn to the surface of WT Hif M10 and PH-deletion Hif M10Δlph mutant. Representative data from one of three separate experiments are shown.

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Malzemeler

Name	Company	Catalog Number	Comments
5 mL polystyrene round-bottom tube	BD Falcon	60819-138	12 × 75 mm style
Bovine Serum Albumins (BSA)	Sigma-Aldrich	A2058	Suitable for cell culture
Flow cytometer	BD Biosciences	651154	Cell analysis grade for research applications
Vitronectin (Vn) from human plasma	Sigma-Aldrich	V8379-50UG	Cell culture grade
E. coli host (E. Coli BL21)	Novagen	69450-3	Protein expression host
FITC-conjugated donkey anti-sheep antibodies	AbD Serotec	STAR88F	Polyclonal
Sheep anti-human Vn antibodies	AbD Serotec	AHP396	Polyclonal