Effects of Complex DNA and MVs with GTF Extracted from Streptococcus mutans on the Oral Biofilm

Abstract

Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases.

The Impacts of Sortase A and the 4′-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis

“…Also prevalent in S. mutans EMVs were glucosyl hydrolases, Gtfs, and Gbps, all of which are well established S. mutans virulence attributors that play a significant role in facilitating biofilm formation (Lemos et al, 2019). GTFs were recently reported to be carried by S. mutans EMVs, and impaired biofilm formation by a gtfBC mutant was restored by exogenous addition of EMVs harvested from broth culture supernatant of the parent strain (Senpuku et al, 2019). Addition of S. mutans EMVs also upregulated biofilm formation by early tooth colonizers including Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus gordonii, Actinomyces naeslundii, and Actinomyces oris, while biofilm formation by other microbial species including Candida albicans, S. salivarius, S. aureus, Streptococcus anginosus, Streptococcus intermedius, and S. pyogenes were not affected (Senpuku et al, 2019).…”

“…GTFs were recently reported to be carried by S. mutans EMVs, and impaired biofilm formation by a gtfBC mutant was restored by exogenous addition of EMVs harvested from broth culture supernatant of the parent strain (Senpuku et al, 2019). Addition of S. mutans EMVs also upregulated biofilm formation by early tooth colonizers including Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus gordonii, Actinomyces naeslundii, and Actinomyces oris, while biofilm formation by other microbial species including Candida albicans, S. salivarius, S. aureus, Streptococcus anginosus, Streptococcus intermedius, and S. pyogenes were not affected (Senpuku et al, 2019). Hence, S. mutans EMVs appear to support biofilm formation not only by S. mutans itself, but also by others that may reside in close proximity in the oral cavity.…”

“…This disparity may stem from differences in growth conditions or lipid detection methodologies, but suggests that the presence of unsaturated lipids in S. mutans membranes is more common than previously recognized. Several studies have now described the utility of EMVs produced by B. anthracis (Rivera et al, 2010), Neisseria meningitidis (Schwechheimer and Kuehn, 2015), S. aureus (Wang et al, 2018), S. pneumoniae (Brown et al, 2015), and S. mutans (Senpuku et al, 2019) as potential delivery vehicles for vaccine antigens. Intranasal immunization of Balb/c mice with S. mutans EMVs in concert with the TLR3 agonist poly(I-C) was an effective method to raise anti-GtfC IgA and IgG antibodies (Nakamura et al, 2020).…”

In vitro studies of DNA condensation by bridging protein in a crowding environment

“…Similarly, the exogenous addition of EVs from Streptococcus mutans to non-biofilm-forming mutants increases oral biofilm formation. 73…”

“…For Gram-negative bacteria, vesiculation would allow dna translocation across at least the OM, and potentially the IM and the physical barrier of PG. It has been found that the dna associated with biofilm formation is not simply random genomic dna, but complex, short fragments, 73 which could be specifically associated with EVs.…”