Mercedes Rivera

CcpA Regulates Central Metabolism and Virulence Gene Expression in Streptococcus mutans

CcpA globally regulates transcription in response to carbohydrate availability in many gram-positive bacteria, but its role in Streptococcus mutans remains enigmatic. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrated that CcpA plays a direct role in carbon catabolite repression (CCR). Subsequently, the expression of 170 genes was shown to be differently expressed (> or = 2-fold) in glucose-grown wild-type (UA159) and CcpA-deficient (TW1) strains (P < or = 0.001). However, there were differences in expression of only 96 genes between UA159 and TW1 when cells were cultivated with the poorly repressing substrate galactose. Interestingly, 90 genes were expressed differently in wild-type S. mutans when glucose- and galactose-grown cells were compared, but the expression of 515 genes was altered in the CcpA-deficient strain in a similar comparison. Overall, our results supported the hypothesis that CcpA has a major role in CCR and regulation of gene expression but revealed that in S. mutans there is a substantial CcpA-independent network that regulates gene expression in response to the carbohydrate source. Based on the genetic studies, biochemical and physiological experiments demonstrated that loss of CcpA impacts the ability of S. mutans to transport and grow on selected sugars. Also, the CcpA-deficient strain displayed an enhanced capacity to produce acid from intracellular stores of polysaccharides, could grow faster at pH 5.5, and could acidify the environment more rapidly and to a greater extent than the parental strain. Thus, CcpA directly modulates the pathogenic potential of S. mutans through global control of gene expression.

PTH stimulates bone formation in mice deficient in Lrp5

Lrp5 deficiency decreases bone formation and results in low bone mass. This study evaluated the bone anabolic response to intermittent PTH treatment in Lrp5‐deficient mice. Our results indicate that Lrp5 is not essential for the stimulatory effect of PTH on cancellous and cortical bone formation.

Polymicrobial Infection with Periodontal Pathogens Specifically Enhances MicroRNA miR-146a in ApoE−/− Mice during Experimental Periodontal Disease

ABSTRACT Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are periodontal pathogens associated with the etiology of adult periodontitis as polymicrobial infections. Recent studies demonstrated that oral infection with P. gingivalis induces both periodontal disease and atherosclerosis in hyperlipidemic and proatherogenic ApoE−/− mice. In this study, we explored the expression of microRNAs (miRNAs) in maxillas (periodontium) and spleens isolated from ApoE−/− mice infected with P. gingivalis, T. denticola, and T. forsythia as a polymicrobial infection. miRNA expression levels, including miRNA miR-146a, and associated mRNA expression levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) were measured in the maxillas and spleens from mice infected with periodontal pathogens and compared to those in the maxillas and spleens from sham-infected controls. Furthermore, in response to these periodontal pathogens (as mono- and polymicrobial heat-killed and live bacteria), human THP-1 monocytes demonstrated similar miRNA expression patterns, including that of miR-146a, in vitro. Strikingly, miR-146a had a negative correlation with TNF-α secretion in vitro, reducing levels of the adaptor kinases IL-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6). Thus, our studies revealed a persistent association of miR-146a expression with these periodontal pathogens, suggesting that miR-146a may directly or indirectly modulate or alter the chronic periodontal pathology induced by these microorganisms.

Active Invasion of Oral and Aortic Tissues by Porphyromonas gingivalis in Mice Causally Links Periodontitis and Atherosclerosis

Atherosclerotic vascular disease is a leading cause of myocardial infarction and cerebrovascular accident, and independent associations with periodontal disease (PD) are reported. PD is caused by polymicrobial infections and aggressive immune responses. Genomic DNA of Porphyromonas gingivalis, the best-studied bacterial pathogen associated with severe PD, is detected within atherosclerotic plaque. We examined causal relationships between chronic P. gingivalis oral infection, PD, and atherosclerosis in hyperlipidemic ApoEnull mice. ApoEnull mice (n = 24) were orally infected with P. gingivalis for 12 and 24 weeks. PD was assessed by standard clinical measurements while the aorta was examined for atherosclerotic lesions and inflammatory markers by array. Systemic inflammatory markers serum amyloid A, nitric oxide, and oxidized low-density lipoprotein were analyzed. P. gingivalis infection elicited specific antibodies and alveolar bone loss. Fluorescent in situ hybridization detected viable P. gingivalis within oral epithelium and aorta, and genomic DNA was detected within systemic organs. Aortic plaque area was significantly increased in P. gingivalis-infected mice at 24 weeks (P<0.01). Aortic RNA and protein arrays indicated a strong Th2 response. Chronic oral infection with P. gingivalis results in a specific immune response, significant increases in oral bone resorption, aortic inflammation, viable bacteria in oral epithelium and aorta, and plaque development.

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoEnull) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoEnull mice.

Role of Porphyromonas gingivalis phosphoserine phosphatase enzyme SerB in inflammation, immune response, and induction of alveolar bone resorption in rats.

ABSTRACT Porphyromonas gingivalis secretes a serine phosphatase enzyme, SerB, upon contact with gingival epithelial cells in vitro. The SerB protein plays a critical role in internalization and survival of the organism in epithelial cells. SerB is also responsible for the inhibition of interleukin-8 (IL-8) secretion from gingival epithelial cells infected with P. gingivalis. This study examined the ability of a P. gingivalis SerB mutant to colonize the oral cavity and induce gingival inflammation, immune responses, and alveolar bone resorption in a rat model of periodontal disease. Both P. gingivalis ATCC 33277 and an isogenic ΔSerB mutant colonized the oral cavities of rats during the 12-week experimental period. Both of the strains induced significant (P < 0.05) systemic levels of immunoglobulin G (IgG) and isotypes IgG1, IgG2a, and IgG2b, indicating the involvement of both T helper type 1 (Th1) and Th2 responses to infection. Both strains induced significantly (P < 0.05) higher levels of alveolar bone resorption in infected rats than in sham-infected control rats. However, horizontal and interproximal alveolar bone resorption induced by the SerB mutant was significantly (P < 0.05) lower than that induced by the parental strain. Rats infected with the ΔSerB mutant exhibited significantly higher levels of apical migration of the junctional epithelium (P < 0.01) and polymorphonuclear neutrophil (PMN) recruitment (P < 0.001) into the gingival tissues than rats infected with the wild type. In conclusion, in a rat model of periodontal disease, the SerB phosphatase of P. gingivalis is required for maximal alveolar bone resorption, and in the absence of SerB, more PMNs are recruited into the gingival tissues.

Role for β1 integrins in cortical osteocytes during acute musculoskeletal disuse

The mammalian skeleton adjusts bone structure and strength in response to changes in mechanical loading, however the molecular and cellular mechanisms governing this process in vivo are unknown. Terminally differentiated osteoblasts, the osteocytes, are presumptive mechanosensory cells for bone, and cell culture studies demonstrate that beta1 integrins participate in mechanical signaling. To determine the role of beta1 integrins in osteoblasts in vivo, we used the Cre-lox system to delete beta1 integrin from cells committed to the osteoblast lineage. While pCol2.3 Cre-mediated recombination was widespread in bones from Colalpha1(I)-Cre+/beta1fl/fl conditional knockout mice (cKO), beta1 integrin protein was depleted from cortical osteocytes, but not from cancellous osteocytes or cells lining bone surfaces in adults. Bones from cKO mice that were normally loaded were similar in structure to WT littermates. However, hindlimb unloading of adult cKO mice for one week intended to cause bone loss (disuse osteopenia), resulted in unexpected, rapid changes in the geometry of cortical bone; hindlimb unloading increased the cross-sectional area, marrow area, and moments of inertia in cKO, but not WT mice. Furthermore, these hindlimb unloading-induced geometric changes in cortical bone of cKO mice resulted in increased whole bone bending stiffness and strength of the femur. Together, these results confirmed the concept that osteocytes are mechanosensory cells and showed beta1 integrins in cortical osteocytes limited changes in cortical geometry in response to disuse, thus providing the first in vivo evidence that beta1 integrins on osteocytes mediate specific aspects of mechanotransduction.

Effects of Basic Fibroblast Growth Factor and a Prostaglandin E2 Receptor Subtype 4 Agonist on Osteoblastogenesis and Adipogenesis in Aged Ovariectomized Rats

bFGF stimulates osteo‐ and adipogenesis concurrently at skeletal sites with red but not with fatty marrow, whereas a PGE2 receptor subtype 4 agonist has bone anabolic effects at both skeletal sites and decreases adipose tissue within red and fatty marrow.

Active Invasion of Porphyromonas gingivalis and Infection-Induced Complement Activation in ApoE-/- Mice Brains

Periodontal disease is a polymicrobial inflammatory disease that leads to chronic systemic inflammation and direct infiltration of bacteria/bacterial components, which may contribute to the development of Alzheimers disease. ApoE-/- mice were orally infected (n = 12) with Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum as mono- and polymicrobial infections. ApoE-/- mice were sacrificed following 12 and 24 weeks of chronic infection. Bacterial genomic DNA was isolated from all brain tissues except for the F. nucleatum mono-infected group. Polymerase chain reaction was performed using universal 16 s rDNA primers and species-specific primer sets for each organism to determine whether the infecting pathogens accessed the brain. Sequencing amplification products confirmed the invasion of bacteria into the brain during infection. The innate immune responses were detected using antibodies against complement activation products of C3 convertase stage and the membrane attack complex. Molecular methods demonstrated that 6 out of 12 ApoE-/- mice brains contained P. gingivalis genomic DNA at 12 weeks (p = 0.006), and 9 out of 12 at 24 weeks of infection (p = 0.0001). Microglia in both infected and control groups demonstrated strong intracellular labeling with C3 and C9, due to on-going biosynthesis. The pyramidal neurons of the hippocampus in 4 out of 12 infected mice brains demonstrated characteristic opsonization with C3 activation fragments (p = 0.032). These results show that the oral pathogen P. gingivalis was able to access the ApoE-/- mice brain and thereby contributed to complement activation with bystander neuronal injury.

Invasion of Oral and Aortic Tissues by Oral Spirochete Treponema denticola in ApoE−/− Mice Causally Links Periodontal Disease and Atherosclerosis

ABSTRACT Treponema denticola is a predominantly subgingival oral spirochete closely associated with periodontal disease and has been detected in atherosclerosis. This study was designed to evaluate causative links between periodontal disease induced by chronic oral T. denticola infection and atherosclerosis in hyperlipidemic ApoE−/− mice. ApoE−/− mice (n = 24) were orally infected with T. denticola ATCC 35404 and were euthanized after 12 and 24 weeks. T. denticola genomic DNA was detected in oral plaque samples, indicating colonization of the oral cavity. Infection elicited significantly (P = 0.0172) higher IgG antibody levels and enhanced intrabony defects than sham infection. T. denticola-infected mice had higher levels of horizontal alveolar bone resorption than sham-infected mice and an associated significant increase in aortic plaque area (P ≤ 0.05). Increased atherosclerotic plaque correlated with reduced serum nitric oxide (NO) levels and increased serum-oxidized low-density lipoprotein (LDL) levels compared to those of sham-infected mice. T. denticola infection altered the expression of genes known to be involved in atherosclerotic development, including the leukocyte/endothelial cell adhesion gene (Thbs4), the connective tissue growth factor gene (Ctgf), and the selectin-E gene (Sele). Fluorescent in situ hybridization (FISH) revealed T. denticola clusters in both gingival and aortic tissue of infected mice. This is the first study examining the potential causative role of chronic T. denticola periodontal infection and vascular atherosclerosis in vivo in hyperlipidemic ApoE−/− mice. T. denticola is closely associated with periodontal disease and the rapid progression of atheroma in ApoE−/− mice. These studies confirm a causal link for active oral T. denticola infection with both atheroma and periodontal disease.