Candida albicans is an opportunistic fungal organism frequently detected in the oral cavity of children with severe early childhood caries (S-ECC). Previous studies suggested the cariogenic potential of C. albicans, in vitro and in vivo, and further demonstrated its synergistic interactions with Streptococcus mutans. In combination, the 2 organisms are associated with higher caries severity in a rodent model. However, it remains unknown whether C. albicans influences the composition and diversity of the entire oral bacterial community to promote S-ECC onset. With 16s rRNA amplicon sequencing, this study analyzed the microbiota of saliva and supragingival plaque from 39 children (21 S-ECC and 18 caries-free [CF]) and 33 mothers (17 S-ECC and 16 CF). The results revealed that the presence of oral C. albicans is associated with a highly acidogenic and acid-tolerant bacterial community in S-ECC, with an increased abundance of plaque Streptococcus (particularly S. mutans) and certain Lactobacillus/Scardovia species and salivary/plaque Veillonella and Prevotella, as well as decreased levels of salivary/plaque Actinomyces. Concurrent with this microbial community assembly, the activity of glucosyltransferases (cariogenic virulence factors secreted by S. mutans) in plaque was significantly elevated when C. albicans was present. Moreover, the oral microbial community composition and diversity differed significantly by disease group (CF vs. S-ECC) and sample source (saliva vs. plaque). Children and mothers within the CF and S-ECC groups shared microbiota composition and diversity, suggesting a strong maternal influence on children's oral microbiota. Altogether, this study underscores the importance of C. albicans in association with the oral bacteriome in the context of S-ECC etiopathogenesis. Further longitudinal studies are warranted to examine how fungal-bacterial interactions modulate the onset and severity of S-ECC, potentially leading to novel anticaries treatments that address fungal contributions.

Streptococcus mutans F-ATPase, the major component of the acid-adaptive response of the organism, is transcriptionally upregulated at low pH. Fusions of the F-ATPase promoter to chloramphenicol acetyltransferase indicated that pH-dependent expression is still observed with a short promoter that contains a domain conserved between streptococcal ATPase operons.During growth at acidic pH values, Streptococcus mutans and S. sanguis both increase production of their F-ATPases (4,5,12). By pumping protons out of cells, the enzymes function to assist in maintaining internal pH values (6,8,13,14). The ATPases from S. mutans and S. sanguis differ in their innate abilities to function in acidic conditions, as evidenced by the pH optima of the enzymes: approximately 6.0 for S. mutans and 7.0 for S. sanguis, with the maximal induction of the S. mutans enzyme occurring at pH 5. Thus, the ATPase is likely a significant reason why the mutans streptococci are able to outcompete S. sanguis during growth in low-pH environments (21) and to cause dental caries (18,24).The present study was undertaken to determine whether control of ATPase production in oral streptococci occurs at the transcriptional level. RNA slot blots, reporter gene fusions, and computer modeling were used to examine the pH-dependent expression of the S. mutans F-ATPase promoter in S. mutans and S. sanguis.(The material presented here was used in partial fulfillment of the Ph.D. requirements of the University of Rochester by W. L. Kuhnert.).RNA samples from cells grown at pH 5 have greater abundance of ATPase ␤-subunit transcript. We first determined whether ATPase-specific mRNA levels differed in RNA samples prepared from cells grown at pH 5 or 7. S. mutans UA159 was grown in continuous culture to steady state at a dilution rate of 0.24 h Ϫ1 , as described previously (10, 15). RNA slot blots hybridized to a ␤-subunit-specific DNA probe (amplified with primers ␤ Fwd and ␤ Rev; Table 1) showed an approximately twofold increase in the abundance of ATPase message in the cells grown at pH 5 (Fig. 1).ATPase operon transcriptional rate is greater at pH 5 than at pH 7. Three reporter gene fusions were constructed using PCR (25), both in multicopy (plasmid-borne) and single-copy (chromosomal) fusions, to determine the transcriptional activity of the operon. These included (i) an intact promoter fragment of the S. mutans GS-5 ATPase operon fused to a cat gene (contained on pDL556/CAT and in S. mutans UR123); (ii) a fragment containing the Ϫ10 and Ϫ35 sequences and promoter sequence that extended only into the predicted inverted DNA repeat proximal to the structural gene (carried on pDL680/CAT and in S. mutans UR124); and (iii) a promoterless cat gene fusion (contained on pDLCAT and in S. mutans UR122). The appropriate clones of promoter fragments were verified by nucleotide sequencing (15, 26) and were subsequently subcloned into pDL278 (16,17), resulting in the constructs listed above (detailed in Fig. 2).Single-copy fusions were created by integrating the promoter-chlo...

Streptococcus mutans -derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ΔlytS and ΔlytT; LTA – ΔdltA and ΔdltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

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