Complementation of the fadA mutation in Fusobacterium nucleatum demonstrates that the surface-exposed adhesin promotes cellular invasion and placental colonization - PubMed (original) (raw)

Complementation of the fadA mutation in Fusobacterium nucleatum demonstrates that the surface-exposed adhesin promotes cellular invasion and placental colonization

Akihiko Ikegami et al. Infect Immun. 2009 Jul.

Abstract

Fusobacterium nucleatum is a gram-negative oral anaerobe implicated in periodontal disease and adverse pregnancy outcome. The organism colonizes the mouse placenta, causing localized infection and inflammation. The mechanism of placental colonization has not been elucidated. Previous studies identified a novel adhesin from F. nucleatum, FadA, as being involved in the attachment and invasion of host cells. The fadA deletion mutant F. nucleatum 12230 US1 was defective in host cell attachment and invasion in vitro, but it also exhibited pleiotropic effects with altered cell morphology and growth rate. In this study, a fadA-complementing clone, F. nucleatum 12230 USF81, was constructed. The expression of FadA on USF81 was confirmed by Western blotting and immunofluorescent labeling. USF81 restored host cell attachment and invasion activities. The ability of F. nucleatum 12230, US1, and USF81 to colonize the mouse placenta was examined. US1 was severely defective in placental colonization compared to the wild type and USF81. Thus, FadA plays an important role in F. nucleatum colonization in vivo. These results also represent the first complementation studies for F. nucleatum. FadA may be a therapeutic target for preventing F. nucleatum colonization of the host.

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Figures

FIG. 1.

FIG. 1.

Construction of fadA_-complementing clone USF81. Suicide plasmid pYH1480 carrying ermF-fadA-catP-ermAM was sonoporated into fadA deletion mutant US1 (Δ_fadA::ermF-ermAM), in which the fadA gene was almost completely replaced by the ermF-ermAM cassette. Through double-crossover homologous recombination in ermF and ermAM, the wild-type fadA-catP fragment was inserted into the chromosome of US1, generating thiamphenicol-resistant, _fadA_-complementing clone USF81.

FIG. 2.

FIG. 2.

Expression of FadA in F. nucleatum 12230, US1, and USF81. (A) Western blotting analysis using MAb 5G11-3G8. The arrow points to FadA detected in F. nucleatum 12230 (_Fn_12230) and USF81. (B) Immunofluorescent labeling of FadA on non-detergent-treated bacteria using MAb 5G11-3G8. The same fields were captured by both light (i) and fluorescent (ii) microscopes using a 40× objective.

FIG. 3.

FIG. 3.

Attachment (A) and invasion (B) of CHO and immortalized human oral keratinocyte OKF6/Tert (Tert) cells by wild-type F. nucleatum 12230, fadA deletion mutant US1, and the _fadA_-complementing clone USF81. The results shown are averages of data from at least four independent experiments, with each experiment performed in triplicate. The standard deviations are expressed as lines above the bars. In all experiments, no significant difference was found between F. nucleatum 12230 and USF81.

FIG. 4.

FIG. 4.

Colonization of F. nucleatum 12230, US1, and USF81 in the mouse placenta. Approximately 2 × 107 CFU of different F. nucleatum strains were injected into each pregnant CF-1 mouse via tail vein on day 16 of gestation. The placentas were harvested 6 or 24 h later. Live bacterial titers in the placentas were determined and expressed as log(CFU/gram tissue). The results shown are averages of data from three to seven mice per strain. The standard deviations are expressed as lines above the bars. *, P < 0.05 compared to F. nucleatum 12230.

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