Adhesion of early oral bacterial colonizers to aged collagen explored by bacterial force spectroscopy

Session

AFM in Life Sciences

Authors

Camila Leiva-Sabadini (4, 5), Dr Nelson Barrera (2), Dr Laurent Bozec (3), Dr Christina Schuh (1), Dr Sebastian Aguayo (4, 5)

Affiliations

1. Centro de Medicina Regenerativa, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo
2. Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile
3. Faculty of Dentistry, University of Toronto
4. School of Dentistry, Faculty of Medicine, Pontificia Universidad Católica de Chile
5. Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences

Keywords

Atomic force microscopy, public health, oral disease, bacterial adhesion, dental caries, biofilms

Abstract text

Despite current efforts, the prevalence of oral pathologies such as dental caries continues to burden healthcare systems worldwide. In this context, the early attachment of relevant oral bacterial colonizers such as Streptococcus mutans and Streptococcus sanguinis to collagen is critical for caries biofilm formation. Particularly, S. mutans adhesion to collagen is implicated in the initiation of dentinal and root caries; therefore, factors promoting the adhesion of this strain to dental surfaces are highly relevant in the clinical setting. Despite our knowledge regarding the influence of aging and glycation on host cell adhesion, the biophysics driving early oral streptococcal adhesion to glycated collagen remains unknown. In this context, molecular and mechanobiological changes in collagen due to aging may play a crucial role in S. mutans biofilm formation and disease progression. Therefore, the objective of this research was to explore the dynamics of the initial adhesion of S. mutans and S. sanguinis to native and glycated type-I collagen with atomic force microscopy (AFM) bacterial force spectroscopy. 

For this, engineered type-I collagen gels were glycated with 10 mM methylglyoxal (MGO) in order to experimentally simulate collagen aging in-vitro. Then, BL-TR400PB iDrive AFM cantilevers were functionalized with living S. mutans or S. sanguinis cells by means of non-destructive poly-L-lysine immobilization. The bacterial probes were subsequently approached to native and glycated collagen surfaces in real-time, with a loading force of 0.2 nN and a dwelling time of 5 seconds employing an Asylum MFP 3D-SA AFM. From the resulting force curves, the adhesion force, number of events, and contour and rupture lengths for each individual single-unbinding event were computed with the Asylum Research AFM software (v. 16.10.211). Data across all experiments were pooled and statistical significances were assessed either with t-tests or Kruskal-Wallis tests according to normality, considering a significance value of p<0.05.

From the resulting experiments, it was observed that the glycation of type-I collagen with MGO increased the adhesion forces and frequency of single-unbinding events between S. mutans and collagen, with the opposite effect being observed for S. sanguinis. The overall adhesion energy between S. mutans functionalized probes and glycated collagen was also higher than in the control condition with native collagen. On the other hand, no changes were observed in rupture or contour lengths among the different surfaces, suggesting that changes in adhesion forces are due to the strengthening of receptor-surface interactions. In summary, these results suggest that collagen glycation by MGO may promote the early adhesion of S. mutans to oral surfaces, and thus play a crucial role in the colonization of collagenous tissues in the context of aging and chronic hyperglycemia. Further work should determine if this increased early attachment of S. mutans to aged collagen also promotes bacterial formation at later stages, as well as its association with the clinical progression of dental caries.

Acknowledgments: This work was supported by ANID FONDECYT #1220804