Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo

Shelburne, Charles E.; Gleason, Raymond M.; Germaine, Gregory R.; Wolff, Larry F.; Mullally, Brian; Coulter, Wilson A.; Lopatin, Dennis E.

doi:10.1016/s0167-7012(01)00362-1

Cited by 34 publications

(39 citation statements)

References 36 publications

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“…Homology searches of the P. gingivalis database did not reveal sigma 32 homologs, and promoter regions of groES and dnaK do not contain CIRCE consensus sequences. Interestingly, the increased expression of groEL, dnaK, and htpG was detected in subgingival plaque samples, the in vivo environment, by QRT-PCR (43). That these genes were also identified in our study suggests that the in vitro coculture system may yield relevant new insights into the first responses of P. gingivalis to the host defenses.…”

Section: Discussionsupporting

confidence: 61%

“…This technology been applied to several systems, including the host response to pathogenic Mycobacterium tuberculosis (50) and the expression of Staphylococcus aureus genes during chronic lung infections in cystic fibrosis patients (16). Furthermore, these methods have been adapted to determine P. gingivalis gene expression in subgingival plaque (43). A more comprehensive monitoring of the host-pathogen dialogue can be obtained with microarray-based expression profiling (8,26).…”

Section: Discussionmentioning

confidence: 99%

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Gene Expression inPorphyromonas gingivalisafter Contact with Human Epithelial Cells

Hosogi¹,

Duncan²

2005

Infect Immun

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Porphyromonas gingivalis, a gram-negative oral anaerobe, is strongly associated with adult periodontitis. The adherence of the organism to host epithelium signals changes in both cell types as bacteria initiate infection and colonization and epithelial cells rally their defenses. We hypothesized that the expression of a defined set of P. gingivalis genes would be consistently up-regulated during infection of HEp-2 human epithelial cells. P. gingivalis genome microarrays were used to compare the gene expression profiles of bacteria that adhered to HEp-2 cells and bacteria that were incubated alone. Genes whose expression was temporally up-regulated included those involved in the oxidative stress response and those encoding heat shock proteins that are essential to maintaining cell viability under adverse conditions. The results suggest that contact with epithelial cells induces in P. gingivalis stress-responsive pathways that promote the survival of the bacterium.Periodontal diseases have long been recognized as bacterial infections, and the presence of Porphyromonas gingivalis is associated with disease activity in adults. The bacterium is a component of subgingival plaque that interfaces with epithelial cells lining the gingival sulcus. It was determined experimentally that cells or fractions of P. gingivalis trigger various events in epithelial cells, including the induction of calcium fluxes (24), the activation of matrix metalloproteinases (9), the upregulation of collagenase and stromelysin expression (10), and the stimulation of interleukin-8 expression in peripheral blood mononuclear cells (25). Of the few reports concerning hostinduced gene expression in P. gingivalis, one study suggested that short contact with gingival epithelial cell monolayers inhibited the secretion of gingipain cysteine proteinases (34), while another reported the induction of Lys-gingipain upon prolonged contact with glutaraldehyde-fixed epithelial cells (1). Most recently, differential display reverse transcription-PCR was used to screen for P. gingivalis genes expressed during internalization in gingival epithelial cells (35). Of the genes identified, those encoding endopeptidase O (pepO), a cationtransporting ATPase, and an ABC transporter were mutated, and subsequent analyses suggested that they played a role in cell invasion.To increase knowledge of host-induced gene expression in P. gingivalis, we determined the profile of transcription of the organism induced by contact with HEp-2 epithelial cells by using a microarray comprising PCR amplicons of all of the open reading frames (ORFs) identified in the genome (33). During the early stages of infection, we observed the expression of genes involved in an oxidative stress response which, by analogy with similar responses in Bacteroides fragilis, might be controlled by OxyR, the peroxide-sensing regulator. Experimental evidence suggested that HEp-2 cells produced reactive oxygen species (ROS) that initiated the response in P. gingivalis. In addition, heat shock genes were expres...

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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Gene Expression inPorphyromonas gingivalisafter Contact with Human Epithelial Cells

Hosogi¹,

Duncan²

2005

Infect Immun

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“…A luxAB-based luciferase reporter system (28) and (quantitative) RT-PCR strategies (4,8,28,38,40) have been employed previously to monitor in situ gene expression in bacteria during residence in their hosts. Both approaches have been applied only to the murine gut and require extensive homogenization of this organ prior to analysis.…”

mentioning

confidence: 99%

Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression inListeria monocytogenes

Bron¹,

Monk²,

Corr³

et al. 2006

Appl Environ Microbiol

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In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes.

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“…Subgingival plaque was collected and immediately placed in a labeled vial containing 500 l of stabilizing buffer to prevent degradation (RNA Protect; Ambion, Austin, TX). After vortexing for 30 s, the samples were stored at 4°C until they were sent to the laboratory for analysis as described previously (42). Colonization of plaque samples was evaluated by real-time PCR as described previously, using primers specific for the species-specific segments of the 16S rRNA genes of P. gingivalis (43).…”

Section: Methodsmentioning

confidence: 99%

“…Colonization of plaque samples was evaluated by real-time PCR as described previously, using primers specific for the species-specific segments of the 16S rRNA genes of P. gingivalis (43). The percentage of the total flora for each species was calculated by dividing the number of target organisms by the total number of bacteria as determined by real-time PCR using 16S rRNA primers that reacted with all bacterial species (42).…”

Section: Methodsmentioning

confidence: 99%

Immunoglobulin G (IgG) Class, but Not IgA or IgM, Antibodies to Peptides of thePorphyromonas gingivalisChaperone HtpG Predict Health in Subjects with Periodontitis by a Fluorescence Enzyme-Linked Immunosorbent Assay

Sweier¹,

Shelburne²,

Giannobile³

et al. 2009

Clin Vaccine Immunol

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Chaperones are molecules found in all cells and are critical in stabilization of synthesized proteins, in repair/removal of defective proteins, and as immunodominant antigens in innate and adaptive immunity. Subjects with gingivitis colonized by the oral pathogen Porphyromonas gingivalis previously demonstrated levels of anti-human chaperone Hsp90 that were highest in individuals with the best oral health. We hypothesized that similar antibodies to pathogen chaperones might be protective in periodontitis. This study examined the relationship between antibodies to P. gingivalis HtpG and clinical statuses of healthy and periodontitissusceptible subjects. We measured the humoral responses (immunoglobulin G [IgG], IgA, and IgM) to peptides of a unique insert (P18) found in Bacteroidaceae HtpG by using a high-throughput, quantitative fluorescence enzyme-linked immunosorbent assay. Indeed, higher levels of IgG class anti-P. gingivalis HtpG P18 peptide (P < 0.05) and P18␣, consisting of the N-terminal 16 amino acids of P18 (P < 0.05), were associated with better oral health; these results were opposite of those found with anti-P. gingivalis whole-cell antibodies and levels of the bacterium in the subgingival biofilm. When we examined the same sera for IgA and IgM class antibodies, we found no significant relationship to subject clinical status. The relationship between anti-P18 levels and clinical populations and individual subjects was found to be improved when we normalized the anti-P18␣ values to those for anti-P18␥ (the central 16 amino acids of P18). That same ratio correlated with the improvement in tissue attachment gain after treatment (P < 0.05). We suggest that anti-P. gingivalis HtpG P18␣ antibodies are protective in periodontal disease and may have prognostic value for guidance of individual patient treatment.

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Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo

Cited by 34 publications

References 36 publications

Gene Expression inPorphyromonas gingivalisafter Contact with Human Epithelial Cells

Gene Expression inPorphyromonas gingivalisafter Contact with Human Epithelial Cells

Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression inListeria monocytogenes

Immunoglobulin G (IgG) Class, but Not IgA or IgM, Antibodies to Peptides of thePorphyromonas gingivalisChaperone HtpG Predict Health in Subjects with Periodontitis by a Fluorescence Enzyme-Linked Immunosorbent Assay

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