Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries

Tooth decay (dental caries) is a widespread human disease caused by microbial biofilms. Streptococcus mutans, a biofilm-former, has been consistently associated with severe childhood caries; however, how this bacterium is spatially organized with other microorganisms in the oral cavity to promote disease remains unknown. Using intact biofilms formed on teeth of toddlers affected by caries, we discovered a unique 3D rotund-shaped architecture composed of multiple species precisely arranged in a corona-like structure with an inner core of S. mutans encompassed by outer layers of other bacteria. This architecture creates localized regions of acidic pH and acute enamel demineralization (caries) in a mixed-species biofilm model on human teeth, suggesting this highly ordered community as the causative agent. Notably, the construction of this architecture was found to be an active process initiated by production of an extracellular scaffold by S. mutans that assembles the corona cell arrangement, encapsulating the pathogen core. In addition, this spatial patterning creates a protective barrier against antimicrobials while increasing bacterial acid fitness associated with the disease-causing state. Our data reveal a precise biogeography in a polymicrobial community associated with human caries that can modulate the pathogen positioning and virulence potential in situ, indicating that micron-scale spatial structure of the microbiome may mediate the function and outcome of host-pathogen interactions

Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries

Tooth decay (dental caries) is a widespread human disease caused by microbial biofilms. Streptococcus mutans, a biofilm-former, has been consistently associated with severe childhood caries; however, how this bacterium is spatially organized with other microorganisms in the oral cavity to promote disease remains unknown. Using intact biofilms formed on teeth of toddlers affected by caries, we discovered a unique 3D rotund-shaped architecture composed of multiple species precisely arranged in a corona-like structure with an inner core of S. mutans encompassed by outer layers of other bacteria. This architecture creates localized regions of acidic pH and acute enamel demineralization (caries) in a mixed-species biofilm model on human teeth, suggesting this highly ordered community as the causative agent. Notably, the construction of this architecture was found to be an active process initiated by production of an extracellular scaffold by S. mutans that assembles the corona cell arrangement, encapsulating the pathogen core. In addition, this spatial patterning creates a protective barrier against antimicrobials while increasing bacterial acid fitness associated with the disease-causing state. Our data reveal a precise biogeography in a polymicrobial community associated with human caries that can modulate the pathogen positioning and virulence potential in situ, indicating that micron-scale spatial structure of the microbiome may mediate the function and outcome of host–pathogen interactions

TLRs activation restores LC3-II expression and inhibits the growth of Mfa1⁺Pg within human MoDCs.

<p><b>A)</b> Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (<i>E. coli</i> LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour. <b>B)</b> Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&2 ligands (<i>E. coli</i> LPS and Pam3csk4) <b>C)</b> The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** <i>p</i><0.001).</p

<i>Porphyromonas gingivalis</i> Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

<div><p>Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of <i>Porphyromonas gingivalis</i> in human monocyte-derived DCs (MoDCs). We employed a panel of <i>P. gingivalis</i> isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+<i>P. gingivalis</i> strains by MoDCs resulted in lower intracellular killing and higher intracellular content of <i>P. gingivalis</i>. Moreover, Mfa1+<i>P. gingivalis</i> was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+<i>P. gingivalis</i> strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, <i>P. gingivalis</i> uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+<i>P. gingivalis</i> promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.</p></div

TLRs activation restores LC3-II expression and inhibits the growth of Mfa1⁺Pg within human MoDCs.

<p><b>A)</b> Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (<i>E. coli</i> LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour. <b>B)</b> Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&2 ligands (<i>E. coli</i> LPS and Pam3csk4) <b>C)</b> The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** <i>p</i><0.001).</p

Mfa1⁺Pg up-regulate the expression of DC-SIGN in human MoDCs.

<p><b>A)</b> DC-SIGN mRNA expression in <i>P. gingivalis</i>-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2^-(ΔΔCt) method. The statistical analysis was performed using the <i>t-test</i>, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* <i>p</i><0.001). <b>B)</b> Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1⁺Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1⁺Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. <b>C)</b> Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1⁺Pg and FimA⁺Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* <i>p</i><0.01).</p

Pearson’s co-localization of Rab5 (red) and P. gingivalis (green) signals.

<p>Pearson’s co-localization of Rab5 (red) and P. gingivalis (green) signals.</p

Formation of double-membrane vesicles in <i>P. gingivalis</i>-infected MoDCs.

<p><b>A)</b> Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1⁺Pg (upper panel 10000x and lower panel 25000x). <b>B)</b> SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated). <b>C)</b> Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1⁺Pg and FimA⁺Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA⁺Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1⁺Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows). <b>D)</b> The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn’s test for multiple comparisons (*<i>p<0.01</i>).</p

Pearson’s co-localization of LAMP1 and <i>P. gingivalis</i> signals.

<p>Pearson’s co-localization of LAMP1 and <i>P. gingivalis</i> signals.</p