Abstract
Biofilm formation is crucial for bacterial community development and host colonization by Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides (CwpB, CwpK), (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system (Asp1, Asp2, SecA2) and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively.
Highlights
Streptococcus salivarius is one of the early colonizers of oral mucosa surfaces in neonates and is a commensal inhabitant of the oral cavity and digestive tract of healthy adults
Twenty-eight representative strains of S. salivarius were screened for auto-aggregation (Figure 1A and Table 1)
We found that addition of Ca2+ led to an increase in auto-aggregation, whereas heat-treatment, addition of EDTA, proteinase K or trypsin led to a reduction in auto-aggregation (Figure 1C), suggesting that proteins are involved in these cell-to-cell interactions
Summary
Streptococcus salivarius is one of the early colonizers of oral mucosa surfaces in neonates and is a commensal inhabitant of the oral cavity and digestive tract of healthy adults. Despite the role of S. salivarius in both oral and digestive tract ecology, the factors that allow this bacterium to become established and maintained in the host environment have not yet been the subject of extensive molecular and genetic analyses. Adhesion processes may include attachment of the bacterial cell to host cells, to components of the extracellular matrix (ECM), to the salivary pellicle on teeth and to soluble factors, as well as to bacterial cells of the same strain (autoaggregation) or genetically distinct species (co-aggregation). S. salivarius is able to form auto-aggregates (Couvigny et al, 2017) and to co-aggregate with numerous oral microorganisms such as the early colonizers Veillonella (Weerkamp and McBride, 1980, 1981; Handley et al, 1987) and Prevotella species (Levesque et al, 2003), the intermediate colonizers Fusobacterium nucleatum (Weerkamp and McBride, 1980; Levesque et al, 2003) and Candida albicans (Nikawa et al, 2001; Levesque et al, 2003), and the late colonizers Tannerella forsythia (Shimotahira et al, 2013) and Porphyromonas gingivalis (Levesque et al, 2003)
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