Jeffrey J. Mans

Distinct transcriptional profiles characterize oral epithelium‐microbiota interactions

Transcriptional profiling, bioinformatics, statistical and ontology tools were used to uncover and dissect genes and pathways of human gingival epithelial cells that are modulated upon interaction with the periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Consistent with their biological and clinical differences, the common core transcriptional response of epithelial cells to both organisms was very limited, and organism‐specific responses predominated. A large number of differentially regulated genes linked to the P53 apoptotic network were found with both organisms, which was consistent with the pro‐apoptotic phenotype observed with A. actinomycetemcomitans and anti‐apoptotic phenotype of P. gingivalis. Furthermore, with A. actinomycetemcomitans, the induction of apoptosis did not appear to be Fas‐ or TNFα‐mediated. Linkage of specific bacterial components to host pathways and networks provided additional insight into the pathogenic process. Comparison of the transcriptional responses of epithelial cells challenged with parental P. gingivalis or with a mutant of P. gingivalis deficient in production of major fimbriae, which are required for optimal invasion, showed major expression differences that reverberated throughout the host cell transcriptome. In contrast, gene ORF859 in A. actinomycetemcomitans, which may play a role in intracellular homeostasis, had a more subtle effect on the transcriptome. These studies help unravel the complex and dynamic interactions between host epithelial cells and endogenous bacteria that can cause opportunistic infections.

Gingival Epithelial Cell Transcriptional Responses to Commensal and Opportunistic Oral Microbial Species

ABSTRACT Transcriptional profiling and ontology tools were utilized to define the biological pathways of gingival epithelial cells modulated by coculture with the oral commensal Streptococcus gordonii and the opportunistic commensal Fusobacterium nucleatum. Overall, F. nucleatum and S. gordonii perturbed the gingival epithelial cell transcriptome much less significantly than the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans perturbed the transcriptome, indicating that there was a greater degree of host adaptation by the commensal species (M. Handfield, J. J. Mans, G. Zheng, M. C. Lopez, S. Mao, A. Progulske-Fox, G. Narasimhan, H. V. Baker, and R. J. Lamont, Cell. Microbiol. 7:811-823, 2005). The biological pathways significantly impacted by F. nucleatum and S. gordonii included the mitogen-activated protein kinase (MAPK) and Toll-like receptor signaling pathways. Differential regulation of GADD45 and DUSP4, key components of the MAPK pathway, was confirmed at the protein level by Western blotting. Modulation of the MAPK pathway is likely to affect host cell proliferation and differentiation. In addition, both the MAPK and Toll-like receptor pathways ultimately converge on cytokine gene expression. An enzyme-linked immunosorbent assay of secreted interleukin-6 (IL-6) and IL-8 demonstrated that F. nucleatum induced production of these cytokines, whereas S. gordonii inhibited secretion from the epithelial cells. Stimulation of secretion of proinflammatory cytokines from epithelial cells may reflect the invasive phenotype of F. nucleatum and contribute to the greater pathogenic potential of F. nucleatum than of S. gordonii.

Role of Porphyromonas gingivalis SerB in Gingival Epithelial Cell Cytoskeletal Remodeling and Cytokine Production

ABSTRACT The SerB protein of Porphyromonas gingivalis is a HAD family serine phosphatase that plays a critical role in entry and survival of the organism in gingival epithelial cells. SerB is secreted by P. gingivalis upon contact with epithelial cells. Here it is shown by microarray analysis that SerB impacts the transcriptional profile of gingival epithelial cells, with pathways involving the actin cytoskeleton and cytokine production among those significantly overpopulated with differentially regulated genes. Consistent with the transcriptional profile, a SerB mutant of P. gingivalis exhibited defective remodeling of actin in epithelial cells. Interaction between gingival epithelial cells and isolated SerB protein resulted in actin rearrangement and an increase in the F/G actin ratio. SerB protein was also required for P. gingivalis to antagonize interleukin-8 accumulation following stimulation of epithelial cells with Fusobacterium nucleatum. SerB is thus capable of modulating host cell signal transduction that impacts the actin cytoskeleton and cytokine production.

The degree of microbiome complexity influences the epithelial response to infection

BackgroundThe human microflora is known to be extremely complex, yet most pathogenesis research is conducted in mono-species models of infection. Consequently, it remains unclear whether the level of complexity of a hosts indigenous flora can affect the virulence potential of pathogenic species. Furthermore, it remains unclear whether the colonization by commensal species affects a host cells response to pathogenic species beyond the direct physical saturation of surface receptors, the sequestration of nutrients, the modulation of the physico-chemical environment in the oral cavity, or the production of bacteriocins. Using oral epithelial cells as a model, we hypothesized that the virulence of pathogenic species may vary depending on the complexity of the flora that interacts with host cells.ResultsThis is the first report that determines the global epithelial transcriptional response to co-culture with defined complex microbiota. In our model, human immortalized gingival keratinocytes (HIGK) were infected with mono- and mixed cultures of commensal and pathogenic species. The global transcriptional response of infected cells was validated and confirmed phenotypically. In our model, commensal species were able to modulate the expression of host genes with a broad diversity of physiological functions and antagonize the effect of pathogenic species at the cellular level. Unexpectedly, the inhibitory effect of commensal species was not correlated with its ability to inhibit adhesion or invasion by pathogenic species.ConclusionStudying the global transcriptome of epithelial cells to single and complex microbial challenges offers clues towards a better understanding of how bacteria-bacteria interactions and bacteria-host interactions impact the overall host response. This work provides evidence that the degree of complexity of a mixed microbiota does influence the transcriptional response to infection of host epithelial cells, and challenges the current dogma regarding the potential versus the actual pathogenicity of bacterial species. These findings support the concept that members of the commensal oral flora have evolved cellular mechanisms that directly modulate the host cells response to pathogenic species and dampen their relative pathogenicity.

Role of the ATM-Checkpoint Kinase 2 Pathway in CDT-Mediated Apoptosis of Gingival Epithelial Cells

The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

Microarray Analysis of Human Epithelial Cell Responses to Bacterial Interaction

Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has--until recently--held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

Human trophoblast responses to Porphyromonas gingivalis infection

Porphyromonas gingivalis is a periodontal pathogen that is also associated with preterm low-birthweight delivery. We investigated the transcriptional responses of human extravillous trophoblasts (HTR-8) to infection with P. gingivalis. Over 2000 genes were differentially regulated in HTR-8 cells by P. gingivalis. In ontology analyses of regulated genes, overpopulated biological pathways included mitogen-activated protein (MAP) kinase signaling and cytokine production. Immunoblots confirmed overexpression of the MAP kinase pathway components MEK3, p38 and Max. Furthermore, P. gingivalis infection induced phosphorylation and activation of MEK3 and p38. Increased production of interleukin (IL)-1beta and IL-8 by HTR-8 cells was demonstrated phenotypically by enzyme-linked immunosorbent assay of HTR-8 cell lysates and culture supernatants. Hence, infection of trophoblasts by P. gingivalis can impact signal transduction pathways and modulate cytokine expression, outcomes that could disrupt the maintenance of pregnancy.

Molecular characterization of Treponema denticola infection-induced bone and soft tissue transcriptional profiles.

Treponema denticola is associated with subgingival biofilms in adult periodontitis and with acute necrotizing ulcerative gingivitis. However, the molecular mechanisms by which T. denticola impacts periodontal inflammation and alveolar bone resorption remain unclear. Here, we examined changes in the host transcriptional profiles during a T. denticola infection using a murine calvarial model of inflammation and bone resorption. T. denticola was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and the calvarial bones were excised. RNA was isolated and analysed for transcript profiling using Murine GeneChip arrays. Following T. denticola infection, 2905 and 1234 genes in the infected calvarial bones and soft tissues, respectively, were differentially expressed (P <or= 0.05). Biological pathways significantly impacted by T. denticola infection in calvarial bone and calvarial tissue included leukocyte transendothelial migration, cell adhesion (immune system) molecules, cell cycle, extracellular matrix-receptor interaction, focal adhesion, B-cell receptor signaling and transforming growth factor-beta signaling pathways resulting in proinflammatory, chemotactic effects, and T-cell stimulation. In conclusion, localized T. denticola infection differentially induces transcription of a broad array of host genes, the profiles of which differed between inflamed calvarial bone and soft tissues.

Polymicrobial periodontal pathogen transcriptomes in calvarial bone and soft tissue

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are consistently associated with adult periodontitis. This study sought to document the host transcriptome to a P. gingivalis, T. denticola, and T.forsythia challenge as a polymicrobial infection using a murine calvarial model of acute inflammation and bone resorption. Mice were infected with P. gingivalis, T. denticola, and T. forsythia over the calvaria, after which the soft tissues and calvarial bones were excised. A Murine GeneChip(®) array analysis of transcript profiles showed that 6997 genes were differentially expressed in calvarial bones (P < 0.05) and 1544 genes were differentially transcribed in the inflamed tissues after the polymicrobial infection. Of these genes, 4476 and 1035 genes in the infected bone and tissues were differentially expressed by upregulation. Biological pathways significantly impacted by the polymicrobial infection in calvarial bone included leukocyte transendothelial migration (LTM), cell adhesion molecules, adherens junction, major histocompatibility complex antigen, extracellular matrix-receptor interaction, and antigen processing and presentation resulting in inflammatory/cytokine/chemokine transcripts stimulation in bone and soft tissue. Intense inflammation and increased activated osteoclasts were observed in calvarias compared with sham-infected controls. Quantitative real-time RT-PCR analysis confirmed that the mRNA level of selected genes corresponded with the microarray expression. The polymicrobial infection regulated several LTM and extracellular membrane pathway genes in a manner distinct from mono-infection with P. gingivalis, T. denticola, or T. forsythia. To our knowledge, this is the first definition of the polymicrobially induced transcriptome in calvarial bone and soft tissue in response to periodontal pathogens.

Cellular and bacterial profiles associated with oral epithelium-microbiota interactions.

In the last several years, the momentum towards utilizing global profiling methods has increased, and many studies have incorporated these approaches in order to gain insights into previously undetected host or microbe responses during the host-microbe encounter. Over 100 studies available on NIH Pubmed have used microarrays or similar technologies to assess the transcriptional basis for observed physiological outcomes. Further, these studies have revealed a high level of transcriptional activity that is not readily observable and, indeed, often phenotypically silent; presumably the cell’s attempt to maintain homeostasis and symbiotic harmony (12, 28). Additionally, proteomic studies which profile the entire arsenal of proteins present in a cell at a given time are now also possible. Each technique has its own advantages and limitations, and the technologies themselves are reviewed elsewhere (3, 10, 16, 19, 20, 30, 34). As also reviewed previously, the epithelium is the first line of defense to human infections initiating at mucosal membranes (4, 7), and several studies have demonstrated the specific and active nature of the epithelial response to microbial encounters. The response of the epithelium to bacterial, viral, fungal and protozoal challenge has yielded significant insights into the role of the epithelium in host defense. In addition to a passive role as a physical barrier, the epithelium actively participates in the recruitment of immune effectors, and can directly fend off microbes via defensins and other innate immune mechanisms. Despite the useful information that can be gained from transcriptional or proteomic approaches that study the epithelium, only a limited number of studies have been performed which are specifically targeted to the oral epithelium. Even fewer are targeted specifically to probe host-pathogen interactions of the oral epithelium, as many global genomic studies focus on oral carcinogenesis. Nonetheless, profiling of the host epithelium and/or the infecting microbe has emerged as a useful reporter of specific interactions. Both in vitro and in vivo studies have been performed which have yielded potential markers for periodontal disease progression, novel targets for therapeutics, and have improved the general understanding of host-pathogen cross talk. This review will highlight some important studies which have been conducted to gain insight into cellular and bacterial transcriptional and proteomic profiles associated with the oral cavity. In general, we will consider both sides of the story; the host perspective and that of the colonizing microbe. In closing, the state of the field will be considered and some suggestions will be made to best utilize existing data and overcome existing challenges to understanding the complex interplay between host and microbe in the oral cavity.