Nanoscale characterization of oral streptococci early-adhesion to crosslinked type-I collagen matrices by atomic force microscopy

Authors

Dr Sebastian Aguayo (1, 2)

Affiliations

1. Dentistry School, Faculty of Medicine, Pontificia Universidad Católica de Chile
2. Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile

Keywords

atomic force microscopy, bacterial adhesion, dental caries, biofilms, Streptococcus mutans

Abstract text

Currently, dental caries remains a highly prevalent disease affecting patients worldwide. Although multifactorial, one of the main drivers behind this pathology is the establishment of a specific biofilm on the surface of the tooth. In dental caries, this biofilm is dominated by Streptococcus mutans, an acid-producing strain that progressively demineralizes the tooth surface. Furthermore, the known ability of S. mutans to bind collagen allows it to penetrate the deeper layers of the tooth and periodontal tissue, from where it can also migrate to remote locations in the body. Collagen, and most importantly type-I collagen, is the most important structural protein in the human body. Type-I collagen suffers alterations such as non-enzymatic crosslinking by the action of glucose and its derivates such as methylglyoxal (MGO), which is relevant during aging as well as in certain diseases such as diabetes mellitus. Despite the well-known effect of collagen crosslinking on host cell attachment, the impact of this process on bacterial adhesion remains poorly understood. Thus, the aim of this work is to elucidate the impact of type-I collagen crosslinking on the early adhesion of S. mutans, by employing atomic force microscopy (AFM) cell force spectroscopy with living bacterial probes. For substrate preparation, 1mg/ml type-I collagen gels were constructed and crosslinked with 10mM MGO for 7 days at 37°C. S. mutans UA159 were grown on BHI agar plates, harvested, and immobilized onto poly-L-lysine coated BL-TR400PB iDrive AFM cantilevers (k=0.1N/m). Subsequently, the living bacterial probes were mounted onto an Asylum MFP 3D-SA (Asylum Research) AFM and probed against collagen substrates under PBS buffer. Force spectroscopy experiments were performed in PBS in iDrive mode with a reduced loading force of 200 pN. Adhesion between S.mutans probes and collagen was recorded for both short (0s) and long (5s) dwelling times, obtaining 50 force curves across 5 random locations (250 force curves per condition). Adhesion energy, rupture length, and rupture force values were extracted using the proprietary Asylum Research AFM software, and data was processed with GraphPad Prism v9. Results demonstrated that S. mutans displays unspecific and specific binding to both control and MGO-crosslinked collagen substrates, which increases at 5s dwelling times. Also, S. mutans shows an increase in adhesion to crosslinked collagen, as well as changes in the frequency and force of single-binding events compared to control collagen. In conclusion, S. mutans early-stage adhesion to collagen at the nanoscale is modulated by matrix crosslinking and may explain differences in biofilm formation and caries development observed in diabetic patients and older people.

References