Characterization of Fusobacterium nucleatum adhesin FadA expression in vitro and in vivo

WANG, YU; TANG, JEN-RUEY; LU, ZIYAO; RUBINSTEIN, ROXANA; PAPAPANOU, PANOS N; HAN, YIPING

Nueva York

Jornada; 60th Birnberg Research Program 2017; 2017

Columbia University

Introduction: Fusobacterium nucleatum (Fn)is a Gram-negative anaerobic oral commensal implicated in various human diseases,including periodontal disease, organ abscesses, pregnancy complications,rheumatoid arthritis, GI disorders and colorectal cancer. It has the ability toadhere to and invade different types of host cells, including human epithelialand endothelial cells. A novel adhesinexpressed by Fn, FadA (for Fusobacteriumadhesin A), is a critical and by far the bestcharacterized virulence factor of Fn.FadA exists in two forms: the non-secreted precursor form (pre-FadA, 15.5kDa)and the secreted mature form (mFadA, 13.6 kDa). Both pre-FadA and mFadAare required to form an active complex, FadAc, for binding and invasion. The preliminary study also showed that serumantibody titers against FadA are increased in patients with periodontitiscompared to periodontally healthy controls, suggesting a role of FadA inperiodontitis. As Fn isconsidered a primarily commensal organism, the question arises how it switchesfrom a commensal to a pathogen. Our central hypothesis is that the commensaland pathogenic states are determined largely through regulation of FadA. Objective: To investigate theregulation of FadA expression and secretion in vitro, andits association with periodontitis in humans.  Materials and methods: F. nucleatum12230 and ATCC 23726 were used for in vitroculture. FadA expression levels were determined by quantitative polymerasechain reaction, western blot and immunohistochemistry. Colon cancer cell line DLD-1 was used for tissue culture attachment and invasion assays. To examine FadA expression in the human oral cavity, subgingivalplaque samples were collected from a group of 12 patients with chronic periodontitisadmitted to the Columbia University College of Dental Medicine PeriodonticsClinic. Patients who have been on antibiotic therapy within three months ofenrollment were excluded. For each patient, two subgingival plaque samples werecollected, with one pooled from the diseased sites (probing depth > 7mm withclinical attachment loss) and another pooled from healthy sites (probing depth< 3 mm without clinical attachment loss).The FadA gene and itsexpression levels were determined. Results & Conclusions:  FadA expression differs between log andstationary phases in planktonic culture, and between shallow and deepperiodontal pockets in humans, respectively. Abundant secreted FadA (sFadA) was observedin the stationary phase and in deep periodontal pockets but not in the logphase or in shallow pockets. Fn instationary phase adheres to and invades host cells more efficiently than in thelog phase. The results suggest that the differential expression of FadA mayaffect Fn virulence and sFadA may play a novel role in Fn interaction with the host and diseasepathogenesis. Discussion:  Futurestudies will examine the mechanism of FadA secretion and the role of sFadA in periodontaldisease.