Fusobacterium nucleatum: a commensal-turned pathogen

Abstract

Fusobacterium nucleatum is an anaerobic oral commensal and a periodontal pathogen associated with a wide spectrum of human diseases. This article reviews its implication in adverse pregnancy outcomes (chorioamnionitis, preterm birth, stillbirth, neonatal sepsis, preeclampsia), GI disorders (colorectal cancer, inflammatory bowel disease, appendicitis), cardiovascular disease, rheumatoid arthritis, respiratory tract infections, Lemierre’s syndrom and Alzheimer’s disease. The virulence mechanisms involved in the diseases are discussed, with a particular emphasis on its colonization, systemic dissemination, and induction of host inflammatory and tumorigenic responses. The FadA adhesin/invasin conserved in F. nucleatum is a key virulence factor and a potential diagnostic marker for F. nucleatum-associated diseases.

With the advancement of microbial detection technologies, an increasing number of previously overlooked microorganisms have been discovered to play important roles in human diseases. Fusobacterium nucleatum, a Gram-negative anaerobe, is such an emerging pathogen that is quickly attracting attention of the medical and research communities. F. nucleatum is ubiquitous in the oral cavity, absent or infrequently detected elsewhere in the body under normal conditions [1,2]. Under disease conditions, however, F. nucleatum is one of the most prevalent species found in extra-oral sites [3]. F. nucleatum is a heterogeneous species with five proposed subspecies (ss), i.e. ss animalis, ss fusiforme, ss nucleatum, ss polymorphum, and ss vincentii, whose prevalence in disease vary [3–6]. This article reviews the infections implicating F. nucleatum, along with the virulence mechanisms involved.

Diseases implicating F. nucleatum

Summarized in Table 1 are diseases in which F. nucleatum has been implicated.

Table 1.

Diseases F. nucleatum associated with.

Diseases		References
Oral Infections	Chronic Periodontitis	[8–11]
	Aggressive Periodontitis	[8,11,12]
	Gingivitis	[8,11,13–15]
	Endodontic Infections	[16–22]
Adverse Pregnancy Outcomes	Chorioamnionitis	[38–40,42]
	Preterm Birth	[39,41–44,46,47]
	Stillbirth	[38]
	Neonatal Sepsis	[37]
	Preeclampsia	[45]
GI Disorders	Colorectal Cancer	[62–71]
	Inflammatory Bowel Disease	[76,77]
	Appendicitis	[78–80]
Other Infections	Atherosclerosis	[84,85,87]
	Cerebral Aneurysm	[86]
	Lemierre’s Syndrome	[81,83]
	Other Respiratory Tract Infections	[81]
	Organ Abscesses	[81]
	Rheumatoid Arthritis	[89]
	Alzheimer’s	[88]

Oral infections

F. nucleatum is one of the most abundant species in the oral cavity, in both diseased and healthy individuals [7–10]. It is implicated in various forms of periodontal diseases including the mild reversible form of gingivitis and the advanced irreversible forms of periodontitis including chronic periodontitis, localized aggressive periodontitis and generalized aggressive periodontitis [8–15] (Table 1). It is also frequently associated with endodontic infections such as pulp necrosis and periapical periodontitis [16–22] (Table 1). The prevalence of F. nucleatum increases with the severity of disease, progression of inflammation and pocket depth [8,14,23]. Among the five subspecies, ss fusiforme and ss vinvcentii are more frequently associated with health while ss nucleatum with disease [24,25]. In addition to the periodontal sites, F. nucleatum is detected in saliva, with its quantities increased in patients with gingivitis and periodontitis, compared to the healthy controls [11,26]. Serum antibody titers to F. nucleatum have been reported to be elevated in diseased patients [27].

The abundance of F. nucleatum is affected by environmental factors. Smoking increases the abundance in both periodontally healthy and diseased individuals [28,29]. Among patients with chronic periodontitis, those with uncontrolled type-2 diabetes have higher levels of F. nucleatum [30].

Animal studies support a causative role of F. nucleatum in periodontal infections. Mono-infection of mice with F. nucleatum induces periodontal bone loss or abscess [31]. When F. nucleatum is co-infected with other oral species, e.g. Tannerella forsythia, Porphyromonas gingivalis and Streptococci, respectively, synergy in virulence is observed as evidenced by enhanced bone loss, abscess, or death [32–36].

Adverse pregnancy outcomes

Adverse pregnancy outcome (APO) is a broad term including preterm labor, chorioamnionitis, preterm premature rupture of membranes, preeclampsia, miscarriage, intrauterine growth retardation, low birth weight, stillbirth, neonatal sepsis, etc. F. nucleatum is one of the most prevalent species and by far the most prevalent oral species implicated in APO [30]. It has been detected in a wide variety of placental and fetal tissues including amniotic fluid, fetal membranes, cord blood, neonatal gastric aspirates, fetal lung and stomach, associated with chorioamnionitis, preeclampsia, preterm birth, stillbirth, and early-onset neonatal sepsis [37–45] (Table 1). A case report of term stillbirth caused by oral F. nucleatum provides the first human evidence that the bacteria originated from the mother’s subgingival plaque and translocated to the placenta and fetus, causing acute inflammation leading to the fetal demise [38]. F. nucleatum has been detected as a predominant species in amniotic fluid and fetal membrane associated with preterm birth [39,43,46,47], and in cord blood associated with early-onset neonatal sepsis [37]. Concurrent detection of F. nucleatum in matching amniotic fluid and cord blood indicates its ability to spread to different placental and fetal compartments [37].

F. nucleatum is frequently detected in amniotic fluid and cord blood by culture-independent methods in cases of idiopathic preterm birth and presumed neonatal sepsis, i.e. the patients display the symptoms of disease but the hospital culture results are negative [37,39]. The prevalence of F. nucleatum detected in cord blood from neonatal sepsis equals or is higher than that of E. coli and Group B Streptococcus, placing F. nucleatum on the same importance scale as these two well-recognized neonatal pathogens [37]. These findings point to the urgent need to update the microbial diagnostic technologies employed by hospital laboratories.

It has been postulated that F. nucleatum translocates from the maternal oral cavity to the intrauterine cavity via hematogenous transmission [48–50]. This hypothesis is supported by results from animal studies [51,52]. Hematogenous injection of F. nucleatum resulted in specific colonization and proliferation of the bacteria in the fetoplacental unit without causing systemic infections. The bacteria colonized initially in the decidua by crossing the endothelium, followed by spread to amniotic fluid, fetus and fetal membrane, mimicking chorioamnionitis, eventually leading to preterm and term fetal death [51]. The pattern and duration of infection, as well as the pathology of the mouse placenta, correspond to those of the stillbirth case described above [38]. The inflammatory responses observed in infected mouse placentas are also consistent with those in humans [51,52]. A recent report that the human placenta harbors a low abundant microbiome closely mimicking the human oral microbiome provides further support for hematogenous transmission [53].

Among the five subspecies, only two have been detected in intrauterine infections, with the overwhelming majority belonging to ss animalis, and an occasional few belonging to ss polymorphum. F. nucleatum is often detected together with other oral species as mixed infections, indicating co-translocation from the oral cavity [37,39,42]. These observations are supported by animal studies in which a variety of cultivated and uncultivated oral species are found to co-translocate to the mouse placenta [54]. The majority of oral species translocated to the murine placenta were oral commensals, supporting the notion that commensals in the oral cavity may become pathogens elsewhere. Accurate identification of species/subspecies involved in APO will make it plausible for early and accurate diagnosis to identify individuals at risk [55].

A large body of literature supports the link between periodontal disease and APO [48]. A few studies conducted in South America, Europe, and U.S. improve birth outcome following repeated periodontal treatment or daily use of antibacterial mouth rinse [56–58]. Unfortunately, several large-scale multi-center intervention studies employing a single-treatment therapy during the second trimester fail to demonstrate efficacy [59–61]. It is possible that the one-time treatment is insufficient for disease resolution, and a daily regimen to control inflammation and oral bacterial load might be more effective [48,49].

GI disorders

The GI disorders discussed here include colorectal cancer (CRC), inflammatory bowel disease (IBD) and appendicitis (Table 1). It is only recently that F. nucleatum has been linked to CRC, yet the field is fast growing. F. nucleatum was first discovered to be enriched in CRC carcinomas and the rectal swabs of CRC patients [62–65]. Subsequent studies report that levels of F. nucleatum are also elevated in adenomas, in stools of patients with adenoma and carcinoma, and associated with stages of colorectal neoplasia development [66–70]. As in APO, F. nucleatum is often detected in conjunction with other oral species, suggesting an oral source of infection [71]. The question arises whether F. nucleatum is a passenger or a driver of colorectal tumorigenesis [72,73]. Rubinstein et al. demonstrate that F. nucleatum stimulates CRC cancer growth by modulating the E-cadherin/β catenin signaling via its unique FadA adheisn (see below). Kostic et al. report that F. nucleatum potentiates colorectal tumorigenesis in Apc^min/+ mice. Together, these studies support a causal role of F. nucleatum in CRC [74,75].

IBD has been recognized as a risk factor for CRC. Thus, it is not surprising that the same microorganisms are implicated in both diseases. F. nucleatum has been detected in colonic biospsies of patients with IBD [76,77]. F. nucleatum strains isolated from inflamed tissues of the IBD patients are more invasive than those from the normal tissues [77]. Several studies have reported association of F. nucleatum in appendicitis [78–80]. Co-occurrence of F. nucleatum with other oral taxa has been observed [80]. The mechanisms of F. nucleatum in IBD and appendicitis have not been elucidated.

Other infections

Fusobacterium nucleatum is associated with a wide spectrum of infections and abscesses including infections of the head and neck (Lemierre’s syndrome, acute and chronic mastoiditis, chronic otitis and sinusitis, tonsillitis, peritonsillar and retropharyngeal abscesses, postanginal cervical lymphadenitis, periodontitis), brain, lungs, abdomen, pelvis, bones, joints, and blood, which has been recently reviewed elsewhere [81,82]. Fusobacterium, especially F. necrophorum and F. nucleatum, is a major cause of the well-known Lemierre’s Syndrome, a rare form of upper airways infection with a life threatening secondary septic thrombophlebitis of internal or external jugular veins, usually developed in previously healthy young adults [81,83].

F. nucleatum has been implicated in cardiovascular diseases (CVD) [3]. It is frequently detected in the atherosclerotic plaques, and is also one of the most common periodontal pathogens detected in ruptured cerebral aneurysm [84–86]. The frequency of detecting F. nucleatum in atherosclerotic plaques and blood vessels is directly related to the severity of periodontal disease [87].

Additional diseases that F. nucleatum has been involved in include rheumatoid arthritis and Alzheimer’s disease [88,89]. Periodontal treatment has been shown to improve clinical outcomes of rheumatoid arthritis [90].

Virulence mechanisms of F. nucleatum

How can an oral commensal be implicated in so many infections within and outside the mouth? The answer lies in several key virulence mechanisms F. nucleatum possesses, which can be broadly classified into two groups and reviewed below: (1) colonization and dissemination, and (2) induction of host responses. The readers can in addition refer to a previous review [82].

Colonization and dissemination

F. nucleatum is an adhesive bacterium. It coaggregates with various microbial species in the oral cavity, playing a key role in dental plaque formation [91]. F. nucleatum encodes several adhesins for interspecies interactions, including Fap2, RadD, and aid1 [92–94]. F. nucleatum also binds to a variety of mammalian cells, i.e. epithelial and endothelial cells, PMNs, monocytes, erythrocytes, fibroblasts, HeLa cells and NK cells, as well as host molecules such as salivary macromolecules, extracellular matrix proteins, human IgG, and cadherins [82,95]. Furthermore, F. nucleatum invades epithelial and endothelial cells [51,96]. Adherence and invasion are essential mechanisms for colonization, dissemination, evasion of host defense, and induction of host responses. Invasion of endothelial cells by F. nucleatum in mouse placenta has been observed [51]. The invasiveness of F. nucleatum varies widely among different strains, and has been shown as directly related to the IBD disease status [77,97].

So far, only one adhesin, FadA, has been identified to bind host cells, and remains to be the best-characterized virulence factor identified from F. nucleatum [82]. FadA exists in two forms, the intact pre-FadA consisting of 129 amino-acid (aa) residues and the secreted mature FadA (mFadA) consisting of 111 aa residues [98]. The crystal structure of mFadA reveals a predominantly alpha-helical hairpin structure, with the monomers linked together in a head-to-tail pattern via a novel leucine-chain motif [98]. Pre-FadA and mFadA form an active complex, FadAc, for host-cell binding and invasion [68,98,99].

FadA is uniquely encoded by two closely related species, F. nucleatum and F. periodonticum, absent in most other species of Fusobacterium [100]. Thus, FadA is a potential diagnostic marker for specific detection of F. nucleatum and F. periodonticum. FadA is more frequently detected in the dental plaque samples from patients with gingivitis and periodontitis than the normal controls [13]. In a proof-of-concept study, the fadA gene level is shown to be step-wise elevated from colon tissues of normal individuals to adenomas, and from adenomas to carcinomas [68]. The fadA levels in the normal tissues adjacent to adenoma and carcinoma are higher than those from individual without tumor or inflammation, indicating a field effect [68].

FadA is not only an adhesin but also an invasin [98]. It is required for binding and invasion of both normal and cancerous host cells, and for colonizing the murine placentas [51,68,82,96,101]. FadA binds to cell-junction molecules, the cadherins. FadA binds to VE-cadherin on the endothelial cells and to E-cadherin on epithelial and colorectal cancer cells [68,101]. Because cadherins are widespread in various tissues and cells, FadA binding to cadherins is likely the reason why it can colonize numerous tissues and body sites. FadA binding to VE-cadherin on endothelial cells causes the latter to migrate from cell-cell junction to intracellular compartments, increasing the permeability of the endothelial layer [101]. Thus, FadA allows both direct invasion into the host cells and pericellular invasion via loosened cell-cell junctions. It is postulated that this is the mechanism employed for systemic dissemination [101]. Furthermore, the increased endothelial permeability allows other bacteria in the vicinity to penetrate through, a likely reason why F. nucleatum is often found in mixed infections at extra-oral sites. It has been shown in vitro that F. nucleatum facilitates both intra-cellular and inter-cellular invasion of other species, such as Streptococcus cristatus and E. coli [101,102].

Induction of host responses

F. nucleatum elicits a variety of host responses [82]. It induces human b-defensin 2 from oral epithelial cells via FAD-I [103], stimulates factors predisposing to atherosclerosis by GroEL [65], and activates lymphocyte apoptosis by Fap2 and RadD [94]. F. nucleatum is a potent stimulator of inflammatory cytokines, IL-6, IL-8, and TNFα [96,104]. Binding of F. nucleatum to NK cells activates inflammatory responses involved in periodontal disease [31]. It is reported that F. nucleatum activates the immune responses through retinoic acid-inducible gene I (RIG-I) [105]. During periodontal health, the pro- and anti-inflammatory factors are maintained under homeostasis. Once disseminated outside the oral cavity and under dysbiosys, F. nucleatum induces exacerbated inflammation thus turning into a pathogen. For example, F. nucleatum stimulates TLR4-mediated inflammatory responses in the placentas of pregnant mice, causing fetal demise [51,52]. Suppression of inflammation protects the fetuses, even in the presence of bacterial colonization [52]. In colorectal cancer cells, F. nucleatum activates not only inflammatory responses, but also oncogenes and Wnt gene expressions, all of which are hallmarks of tumorigenesis [68]. F. nucleatum modulates the tumor-immune microenvironment and selectively expands myeloid cells in Apc^min/+ mice [66].

FadA plays a key role in induction of the tumorigenic responses. A synthetic peptide that prevents FadA from binding to E-cadherin blocks tumorigenic responses [68]. Induction of inflammatory responses requires internalization of FadA in the cancer cells while activation of Wnt and oncogenes does not [68]. It is possible that FadA interacts with intracellular components, such as RIG-I, to activate the inflammatory responses.

Concluding remarks

As a fastidious anaerobe, cultivation of F. nucleatum has been difficult. Hence, although cultivable, F. nucleatum is frequently missed in routine culture employed by hospital laboratories. The recent discovery of this opportunistic commensal in a wide spectrum of human disease is due largely to the employment of culture-independent methods. In addition, not all subtypes of F. nucleatum are equally prevalent in diseases. Thus, it is crucial to update microbial detection technologies in clinical practice for accurate diagnosis of disease and for identification of individuals at risk. The involvement of F. nucleatum in some of the diseases discussed above is still at the stage of association, with no established causal roles. Further more, since F. nucleatum is prevalent in periodontal disease, the link between periodontal health and these human diseases needs to be explored. It is hopeful that with an increasing attention on this emerging commensal-turned pathogen, an output of research findings on the pathogenesis mechanisms of F. nucleatum, its detection, and the connection between oral health and various human diseases can be anticipated in the coming years.

Highlights.

• F. nucleatum is an oral commensal implicated in oral infections, adverse pregnancy outcomes, GI disorders, and various other human diseases.

• F. nucleatum can disseminate systemically colonizing different body sites.

• F. nucleatum elicit a spectrum of host responses including inflammation.

• The FadA adhesin/invasin of F. nucleatum binds to cadherins and is a key virulence factor.