Detection and characterization of autoagglutination activity by Campylobacter jejuni - PubMed (original) (raw)

Detection and characterization of autoagglutination activity by Campylobacter jejuni

N Misawa et al. Infect Immun. 2000 Nov.

Free PMC article

Abstract

In several gram-negative bacterial pathogens, autoagglutination (AAG) activity is a marker for interaction with host cells and virulence. Campylobacter jejuni strains also show AAG, but this property varies considerably among strains. To examine the characteristics of C. jejuni AAG, we developed a quantitative in vitro assay. For strain 81-176, which shows high AAG, activity was optimal for cells grown for < or = 24 h, was independent of growth temperature, and was best measured for cells suspended in phosphate-buffered saline at 25 degrees C for 24 h. AAG activity was heat labile and was abolished by pronase or acid-glycine (pH 2.2) treatment but not by lipase, DNase, or sodium metaperiodate. Strain 4182 has low AAG activity, but extraction with water increased AAG, suggesting the loss of an inhibitor. Strain 6960 has weak AAG with no effect due to water extraction. Our study with clinical isolates suggests that C. jejuni strains may be grouped into three AAG phenotypes. A variant derived from strain 81116 that is flagellate but immotile showed the strong AAG exhibited by the parent strain, suggesting that motility per se is not necessary for the AAG activity. AAG correlated with both bacterial hydrophobicity and adherence to INT407 cells. Mutants which lack flagella (flaA, flaB, and flbA) or common cell surface antigen (peb1A) were constructed in strain 81-176 by natural transformation-mediated allelic exchange. Both AAG activity and bacterial hydrophobicity were abolished in the aflagellate mutants but not the peb1A mutant. In total, these findings indicate that C. jejuni AAG is highly associated with flagellar expression.

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Figures

FIG. 1

FIG. 1

Effect of assay conditions on the AAG activity of_C. jejuni_ strains 81-176, 4182, and 6960. (A) Duration of incubation time. (B) Incubation temperatures. (C) Diluent composition. (D) Age of the culture on TSAS plates. Each assay was conducted by suspending the bacterial cells in PBS (except for panel C), at 25°C (except for panel B) after a 24-h incubation (except for panel A), and the measuring _A_600. Bacterial cells that strongly agglutinate do not remain in the aqueous phase, and_A_600 diminishes.

FIG. 2

FIG. 2

Autoagglutination of cells of _C. jejuni_strains 81-176, 4182, and 6960 after sequential extraction with DW. The AAG assay was conducted in PBS at 25°C after 24-h incubation.

FIG. 3

FIG. 3

Relationship between AAG activity and bacterial hydrophobicity, measured as minimum ammonium sulfate concentration permitting aggregation among 22 clinical C. jejuni isolates. Data are shown for cells before (A) and after (B) extraction with DW. For preextraction, r = 0.909 and P < 0.001, and for postextraction, r = 0.926 and_P_ < 0.001. The circles indicate three phenotypes: ●, substantially strong AAG and hydrophobicity (e.g., strain 81-176); ○, weak AAG and hydrophobicity, which increases after water extraction of cells (e.g., strain 4182); formula image, substantially weak AAG and hydrophobicity, not affected by water extraction (e.g., strain 6960).

FIG. 4

FIG. 4

Phenotypic characterization of the wild-type C. jejuni strain (81-176), the flaAflaB isogenic mutant (strain 98-248), the flbA isogenic mutant (strain 98-255), and the peb1A isogenic mutant (strain 98-311). (A) Motility analysis by semisolid agar assay. (B) Western blotting analysis of whole-cell extracts with rabbit polyclonal anti-flagellin antibody. Fla indicates the flagellin band present in strains 81-176 and 98-311 but absent from strains 98-248 and 98-255 as expected.

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