Ameloblasts are ectoderm-derived cells that produce an extracellular enamel matrix that mineralizes to form enamel. The development and use of immortalized cell lines, with a stable phenotype, is an important contribution to biological studies as it allows for the investigation of molecular activities without the continuous need for animals. In this study we compare the expression profiles of enamel-specific genes in two mouse derived ameloblast-like cell lines: LS8 and ALC cells. Quantitative PCR analysis indicates that, relative to each other, LS8 cells express greater mRNA levels for genes that define secretory-stage activities (Amelx, Ambn, Enam, and Mmp20), while ALC express greater mRNA levels for genes that define maturation-stage activities (Odam and Klk4). Western blot analyses show that Amelx, Ambn, and Odam proteins are detectable in ALC, but not LS8 cells. Unstimulated ALC cells form calcified nodules, while LS8 cells do not. These data provide greater insight as to the suitability of both cell lines to contribute to biological studies on enamel formation and biomineralization, and highlight some of the strengths and weaknesses when relying on enamel epithelial organ-derived cell lines to study molecular activities of amelogenesis.

Summary Treponema denticola levels in the gingival crevice become elevated as periodontal disease develops. Oral treponemes may account for as much as 40% of the total bacterial population in the periodontal pocket. The stimuli that trigger enhanced growth of T. denticola, and the mechanisms associated with the transmission of these signals, remain to be defined. We hypothesize that the T. denticola open reading frames tde1970 (histidine kinase) and tde1969 (response regulator) constitute a functional two‐component regulatory system that regulates, at least in part, responses to the changing environmental conditions associated with the development of periodontal disease. The results presented demonstrate that tde1970 and tde1969 are conserved, universal among T. denticola isolates and transcribed as part of a seven‐gene operon in a growth‐phase‐dependent manner. tde1970 undergoes autophosphorylation and transfers phosphate to tde1969. Henceforth, the proteins encoded by these open reading frames are designated as Hpk2 and Rrp2 respectively. Hpk2 autophosphorylation kinetics were influenced by environmental conditions and by the presence or absence of a PAS domain. It can be concluded that Hpk2 and Rrp2 constitute a functional two‐component system that contributes to environmental sensing.

In the healthy subgingiva, oral treponemes account for a small percentage of the total bacteria. However, in diseased periodontal pockets, treponemes thrive and become a dominant component of the bacterial population. Oral treponemes are uniquely adept at capitalizing on the environmental conditions that develop with periodontal disease. The molecular basis of adaptive responses of oral treponemes is just beginning to be investigated and defined. The completion of several treponeme genome sequences and the characterization of global regulatory systems provide an important starting point in the analysis of signaling and adaptive responses. In this review, we discuss existing literature focused on the genetic regulatory mechanisms of Treponema denticola and present an overview of the possible roles of regulatory proteins identified through genome analyses. This information provides insight into the possible molecular mechanisms utilized by oral spirochetes to survive in the periodontal pocket and transition from a minor to a dominant organism.

The classic organization by Socransky and coworkers categorized the oral bacteria of the subgingival plaque into different complexes. Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia are grouped into the red complex that is highly correlated with periodontal disease. Socransky's work closely associates red with orange complex species such as Fusobacterium nucleatum and Prevotella intermedia but not with members of the other complexes. While the relationship between species contained by these complexes is in part supported by their ability to physically attach to each other, the physiological consequences of these interactions and associations are less clear. In this study, we employed T. denticola as a model organism to analyze contact-dependent responses to interactions with species belonging to the same complex (P. gingivalis and T. forsythia), the closely associated orange complex (using F. nucleatum and P. intermedia as representatives) and the unconnected yellow complex (using Streptococcus sanguinis and S. gordonii as representatives). RNA was extracted from T. denticola alone as well as after pairwise co-incubation for 5 hrs with representatives of the different complexes, and the respective gene expression profiles were determined using microarrays. Numerous genes related to motility, metabolism, transport, outer membrane and hypothetical proteins were differentially regulated in T. denticola in the presence of the tested partner species. Further analysis revealed a significant overlap in the affected genes and we identified a general response to the presence of other species, those specific to two of the three complexes as well as individual complexes. Most interestingly, many predicted major antigens (e.g. flagella, Msp, CTLP) were suppressed in responses that included red complex species indicating that the presence of the most closely associated species induces immune-evasive strategies. In summary, the data presented here provide an in-depth understanding of the transcriptional responses triggered by contact-dependent interactions between microorganisms inhabiting the periodontal pocket.

Treponema denticolais an oral spirochete and periopathogen that transitions from low abundance in healthy subgingival crevices to high abundance in periodontal pockets. The T. denticola response regulator AtcR harbors the relatively rare, LytTR DNA binding domain. LytTR domain containing response regulators control critical transcriptional responses required for environmental adaptation. Using a multi-step bioinformatics approach, 26 strong lytTR recognition motifs were identified in the genome of T. denticola strain 35405. Electrophoretic mobility shift assays (EMSA) demonstrated that AtcR binds to these recogniation motifs. High specificity-high affinity complexes formed with phosphorylated AtcR. The LytTR recognition sequences were found to exist in three distinct promoter architectures designated as LytTR1, LytTR2 and LytTR3 promoters. LytTR1 and LytTR2 promoters harbor σ54 binding sites. The functional diversity of the proteins encoded by the putative AtcR regulon suggests that AtcR sits at the top of a regulatory cascade that plays a central role in facilitating T. denticola’s ability to adapt to changing environmental conditions and thrive in periodontal pockets.