Identification and localization of proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms

Background:To successfully colonize the oral cavity, bacteria must adhere directly or indirectly to the oral surfaces available. Fusobacterium nucleatum plays an important role in the development of the oral biofilm community due to its broad adhesion capabilities, serving as a bridge between the members of the oral biofilm community that cannot be directly joined together. The purpose of this study was to identify and localize the proteins associated with the formation of biofilms of Streptococcus gordonii and F. nucleatum . Methods: Multispecies biofilms were identified by amplification of the srtA and radD genes by real-time PCR. Biofilm cells cultured with sucrose were counted. The protein concentrations in the membrane and cytoplasmic fractions were quantified by western blot. Results: The proteins HSP40 and GAPDH were detected in the cytoplasmic fraction of biofilm and F. nucleatum , respectively. The available anti-GAPDH antibody is specific for GAPDH produced by F. nucleatum , which indicated the coaggregation of F. nucleatum on S. gordonii . Conclusions: HSP40 was only detected in the cytoplasmic fraction of the biofilms, making it one of the essential proteins for adherence. This complex set of interactions could have critical implications for the formation and maturation of oral biofilms in vivo and could provide clues to the mechanism behind the distribution of organisms within the human oral cavity.


Background
The bacterial species of the human oral cavity depend on their ability to bind to surfaces or to each other for colonization and persistence in this nutrient ecological niche.
Therefore, proteins involved in adherence are important components that allow microorganisms to form and reside in complex oral biofilms, in which different groups of 3 bacteria perform specific functions. Although microbial interactions within these biofilms trigger important physiological changes in the associated species, including virulence characteristics, the physical union through specific adhesins is a key element for the successful initiation of surface colonization and biofilm integration [1,2].
Species of the genus Fusobacterium have been linked to a wide variety of microbial species and are considered important for the formation and architecture of biofilms.
Fusobacteria integrate into biofilms by binding to early colonists attached to the surface, such as streptococci and actinomycetes. In addition, fusobacteria recruit other bacterial species, including early colonizers and important periodontal pathogens that cannot directly attach to surfaces. This characteristic allows them to promote changes in the microbial community and impacts their pathogenesis [2].
Culturable oral fusobacteria are predominantly F. periodonticum and F. nucleatum. While F. periodonticum encompasses only one species, F. nucleatum includes five subspecies: nucleatum, polymorphum, fusiforme, animalis, and vincentii. This group of microorganisms thrives not only in subgingival environments [3,4] but also in the supragingival plaque [5]. Streptococci are the most common early colonizers and constitute the main binding partner for the recruitment of fusobacteria in oral biofilms [6].
Bacterial interspecies interactions mediated by adherence are important elements in the formation of oral biofilms. These interactions often occur at a species-specific level, which could determine the health or disease association of a biofilm community. Among the key actors involved in these processes are the fusobacteria that have been recognized for their ability to interact with numerous bacterial groups.
The oral cavity is a great model system for studying polymicrobial interactions since it is home to more than 600 different recognized species of bacteria, most of which are considered to be commensal bacteria [7-10]. Microorganisms in oral biofilms have been 4 categorized into early and late colonizers. The first colonizing species are mainly grampositive, capable of adhering directly to the surface of the tooth and forming the basal layers of the oral biofilm [11][12][13]. Late colonizers are mainly gram-negative bacteria, including certain periodontal pathogens such as Treponema denticola, Tannerella forsythia,and Porphyromonas gingivalis,as well as other bacteria within the oral biofilm, forming a complex network of direct or indirect interactions. The spatial distribution of different bacterial species is important in the formation and architecture of oral biofilms.
Many of the known oral bacterial species do not directly interact with each other; instead, they interact indirectly through their mutual union with F. nucleatum [14].
F. nucleatum is a gram-negative fusiform anaerobic bacterium that has been associated with periodontal disease and a number of systemic diseases. It is considered a "bridge organism" due to its ability to form a colonization bridge between species that do not interact directly, thus playing an integral role in the formation of a mature dental plaque.
The physical attachment between the interacting species is mediated by specific cellular adhesion proteins in their outer membranes [15]. For example, proteins of the SrtA family that anchor to the cell wall have been identified, such as SspA/SspB of S. gordonii, which allow interactions with other streptococci and other oral species, including Porphyromonas gingivalis, Actinomyces, and Candida [16][17][18][19]. Egland et al. [20] showed that the SspA/SspB homologue of S. mutans binds to the host's saliva proteins; the host's matrix proteins, including type I collagen, fibronectin, laminin, and keratin; and serum components such as fibrinogen. Certain strains of actinomycetes can be recognized by the expression of the SpaP protein in S. gordonii along with CshA, a protein typically not present in S. mutans [21].
Recently, fusobacterial interactions with Streptococcus, an important oral carcinogenic pathogen, have been described, but most of the studies focussed on binding to non-5 mutant streptococci, and paired specific adhesins have not yet been identified [22][23][24][25].
The purpose of this study was to identify the proteins linked to the adhesion and coaggregation of microorganisms of the species F. nucleatum and S. gordonii. In this way, possible targets for future therapies that block the incorporation of pathogenic bacteria can be found and can be used as the first biomarkers of oral diseases.

Results
The protein profile of S. gordonii and F. nucleatum from individual cultures run in onedimensional electrophoresis revealed that some proteins were only found in S. gordonii and not in F. nucleatum, and vice versa ( Figure 5, red and yellow arrows). Ct and inverseproportional Ct values determined for the exposed S. gordonii and F. nucleatum biofilms. (table 1) The cytoplasmic protein profile of the biofilms harvested after 1, 4, 7, and 10 days of culture kept constant over time and was similar to the profile of the individual culture of S. gordonii. However, a higher protein load was observed between 50 and 37 kDa ( Figure   6), suggesting the presence of F. nucleatum proteins. On the other hand, no drastic 9 changes were observed in the production of any particular protein from either S. gordonii or F. nucleatum.
Through the detection of GAPDH using a specific antibody, it was determined that the antibody only detected an epitope that is found in GAPDH of F. nucleatum and not of S. gordonii (Figure 7a). Its molecular weight ranged between 50 and 37 kDa. In biofilms, on days 1 and 4 the detection was quite faint compared to at 7 and 10 days (Figure 7b), confirming that the adherence of F. nucleatum on S. gordonii was gradual and definitive at 7 days of culture.
The protein HSP40 was not detected in individual cultures (Figure 8a), but it was detected in biofilms after 7 and 10 days of culture, its molecular weight ranging from 50 to 37 kDa (Figure 8b), indicating that protein is involved in coaggregation and therefore in biofilm formation.
However, the literature on this subject is still scarce [18,37,38]. In this study, we sought to identify proteins associated with the formation of biofilms and whether these proteins are found in the membranes or in the cytoplasm of F. nucleatum and S. gordonii biofilms.
The role of proteins in the adherence and coaggregation of microorganisms in biofilms has been demonstrated in two studies [39,40] showing the overexpression of biofilm growthrelated proteins such as the ATP-binding cassette [41,42]. These membrane proteins are fundamental to many biological processes, such as cell division, control of cell volume, and control of which substances pass through the cell membrane. These results may be relevant because of the role of these proteins in the bacterial resistance to antibiotics [42][43][44][45][46].
A group of proteins that were mostly overexpressed in the biofilm were proteins of unknown function called hypothetical proteins [47]. These data are in line with what has been described by other authors [48,49]. The analysis of these proteins and of their roles in biofilm can provide information on the role of this bacterium in this form of growth [50].
Another protein that is overexpressed in biofilm and may be of interest is NusG [51]. This protein can be used as a marker of F. nucleatum [52] and may be relevant for the development of new diagnostic tools [52,53] not only for periodontitis but also for other diseases associated with the presence of the two microorganisms studied in this paper.

Conclusions
The proteins HSP40 and GAPDH were detected in the cytoplasmic fraction of the biofilm and in F. nucleatum, respectively. The available anti-GAPDH antibody is specific for GAPDH

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.   Figure 1 19 Electrophoresis using srtA and radD from F. nucleatum and S. gordonii.

Figure 2
Tm curve of radD