Effect of salivary agglutination on oral streptococcal clearance by human polymorphonuclear neutrophil granulocytes

Abstract

Salivary agglutination is an important host defense mechanism to aggregate oral commensal bacteria as well as invading pathogens. Saliva flow and subsequent swallowing more easily clear aggregated bacteria compared with single cells. Phagocytic clearance of bacteria through polymorphonuclear neutrophil granulocytes also seems to increase to a certain extent with the size of bacterial aggregates. To determine a connection between salivary agglutination and the host innate immune response by phagocytosis, an in vitro agglutination assay was developed reproducing the average size of salivary bacterial aggregates. Using the oral commensal Streptococcus gordonii as a model organism, the effect of salivary agglutination on phagocytic clearance through polymorphonuclear neutrophil granulocytes was investigated. Here we describe how salivary aggregates of S. gordonii are readily cleared through phagocytosis, whereas single bacterial cells showed a significant delay in being phagocytosed and killed. Furthermore, before phagocytosis the polymorphonuclear neutrophil granulocytes were able to induce a specific de-aggregation, which was dependent on serine protease activity. The data presented suggest that salivary agglutination of bacterial cells leads to an ideal size for recognition by polymorphonuclear neutrophil granulocytes. As a first line of defense, these phagocytic cells are able to recognize the aggregates and de-aggregate them via serine proteases to a more manageable size for efficient phagocytosis and subsequent killing in the phagolysosome. This observed mechanism not only prevents the rapid spreading of oral bacterial cells while entering the bloodstream but would also avoid degranulation of involved polymorphonuclear neutrophil granulocytes, so preventing collateral damage to nearby tissue.

Keywords: Streptococcus gordonii; innate immunity; polymorphonuclear neutrophil granulocytes; salivary agglutination.

FIG 1

Bacterial aggregates in fresh human saliva. Morphology and viability of natural planktonic bacterial aggregates in human saliva. A) Representative phase-contrast picture. B) Merged epifluorescence picture [(blue channel = Alexa Fluor 350 WGA (staining N-acetyl-D-glucosamine and sialic acid on the cell surface of bacteria and eukaryotic cells), red channel = propidium iodide (PI) (general DNA stain to evaluate cell viability, PI can only penetrate compromised membranes), green channel = SYTO 9 (for general DNA staining)]. The close-up areas depict roughly spherical aggregates with an average diameter of approximately 20 μm. Scalebar represents a distance of 20μm. C) Rod and spherical shaped bacterial cells associated with buccal epithelial cells. D) Range of randomly selected salivary aggregates in freshly prepared saliva 1h after regular teeth brushing (n=25).

FIG 2

Saliva induced bacterial agglutination. Observation of saliva induced cellular agglutination kinetics and the resulting culture morphology using 5×10⁸ cfu S. gordonii DL1. A) Decrease in detectable colony forming units. Ultrasonic treated culture aliquots are marked with squares, untreated aliquots are marked with circles. Data represent averages and standard deviations from three independent experiments (*p < 0.004). B) Distribution of aggregate diameter at different timepoints during saliva incubation and after ultrasonic treatment. Diagram shows 25 randomly chosen bacterial aggregates from one representative experiment (dots) and calculated average (bar). C) Representative microscopic phase contrast pictures of average bacterial aggregate size in saliva at different timepoints and after ultrasonic treatment. Scalebar represents a distance of 20μm.

FIG 3

Influence of saliva induced cell agglutination on bacterial appearance in human blood. A) Survival of saliva induced aggregates (circles) and ultrasonic disrupted aggregates (squares) of 2×10⁶ cfu S. gordonii DL1 in human plasma (dashed lines) and whole human blood (solid lines). Data represent averages and standard deviations from three independent experiments (*p < 0.04). B) Influence of low-power ultrasonic treatment on bacterial surface protein composition after incubation in PBS, human plasma, and human saliva followed by plasma incubation.

FIG 4

Influence of saliva induced cell agglutination on phagocytosis. A) Survival of saliva induced aggregates (circles) and ultrasonic disrupted aggregates (squares) of 2×10⁶ cfu S. gordonii DL1 in the presence (solid lines) and absence (dashed lines) of 2×10⁶ granulocytes in RPMI with 10% FCS. B) Corresponding data for determination of vital intracellular bacteria. Data represent averages and standard deviations from three independent experiments (*p < 0.01).

FIG 5

Granulocyte protease dependent deaggregation. Observation of granule-component induced deaggregation kinetics and resulting macroscopic culture morphology using preformed aggregates of 5×10⁸ cfu S. gordonii DL1. A) Increase of detectable colony forming units of preformed bacterial aggregates during incubation with the content of primary granules (squares), primary and azurophilic granules (circles), as well as primary and azurophilic granules in combination with AEBSF (triangles). Data represent averages and standard deviations from three independent experiments (* p < 0.05). B) Representative photographic pictures of macroscopic visible bacterial aggregates in 2ml reaction-tubes.

FIG 6

Determination of granulocyte-protease specificity. Observation of granulocyte-protease dependent deaggregation of preformed aggregates of 5×10⁸ cfu S. gordonii DL1 in the presence of different protease-inhibitor combinations. A) Increase of detectable colony forming units of preformed bacterial aggregates after 60min of incubation. Data represent averages and standard deviations from three independent experiments (* p < 0.03). B) Representative photographic pictures of macroscopic visible bacterial aggregates in 2ml reaction-tubes.

FIG 7

Influence of granulocyte-protease dependent deaggregation on phagocytosis and killing. A) Survival of saliva induced aggregates of 2×10⁶ cfu S. gordonii DL1 with (triangles) and without (circles) AEBSF in the presence (solid lines) and absence (dashed lines) of 2×10⁶ granulocytes in RPMI with 10% FCS. B) Corresponding data for determination of intracellular bacteria. Data represent averages and standard deviations from three independent experiments (* p < 0.04).