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Microbial biofilms promote pathogenesis by disguising antigens, facilitating immune evasion, providing protection against antibiotics and other antimicrobials and, generally, fostering survival and persistence. Environmental fluxes are known to influence biofilm formation and composition, with recent data suggesting that tobacco and tobacco‐derived stimuli are particularly important mediators of biofilm initiation and development in vitro and determinants of polymicrobial communities in vivo. The evidence for tobacco‐augmented biofilm formation by oral bacteria, tobacco‐induced oral dysbiosis, tobacco‐resistance strategies, and bacterial physiology is summarized herein. A general overview is provided alongside specific insights gained through studies of the model and archetypal, anaerobic, Gram‐negative oral pathobiont, Porphyromonas gingivalis. [ABSTRACT FROM AUTHOR]

Aim: The use of cannabis, which contains multiple antimicrobials, may be a risk factor for periodontitis. We hypothesized that multiple oral spirochetes would be phytocannabinoid‐resistant and that cannabidiol (CBD) would act as an environmental stressor to which Treponema denticola would respond transcriptionally, thereby providing first insights into spirochetal survival strategies. Materials and Methods: Oral spirochete growth was monitored spectrophotometrically in the presence and absence of physiologically relevant phytocannabinoid doses, the transcriptional response to phytocannabinoid exposure determined by RNAseq, specific gene activity fluxes verified using qRT‐PCR and orthologues among fully sequenced oral spirochetes identified. Results: Multiple strains of oral treponemes were resistant to CBD (0.1–10 μg/mL), while T. denticola ATCC 35405 was resistant to all phytocannabinoids tested (CBD, cannabinol [CBN], tetrahydrocannabinol [THC]). A total of 392 T. denticola ATCC 35405 genes were found to be CBD‐responsive by RNAseq. A selected subset of these genes was independently verified by qRT‐PCR. Genes found to be differentially activated by both methods included several involved in transcriptional regulation and toxin control. Suppressed genes included several involved in chemotaxis and proteolysis. Conclusions: Oral spirochetes, unlike some other periodontal bacteria, are resistant to physiological doses of phytocannabinoids. Investigation of CBD‐induced transcriptomic changes provided insight into the resistance mechanisms of this important periodontal pathogen. These findings should be considered in the context of the reported enhanced susceptibility to periodontitis in cannabis users. [ABSTRACT FROM AUTHOR]

Aim: Filifactor alocis has recently emerged as a periodontal pathobiont that appears to thrive in the oral cavity of smokers. We hypothesized that identification of smoke‐responsive F. alocis genes would provide insight into adaptive strategies and that cigarette smoke would enhance F. alocis pathogenesis in vivo. Materials and Methods: F. alocis was grown in vitro and cigarette smoke extract‐responsive genes determined by RNAseq. Mice were exposed, or not, to mainstream 1R6F research cigarette smoke and infected with F. alocis, or not, in an acute ligature model of periodontitis. Key clinical, infectious, and immune data were collected. Results: In culture, F. alocis growth was unaffected by smoke conditioning and only a small number of genes were specifically regulated by smoke exposure. Reduced murine mass, differences in F. alocis‐cognizant antibody production, and altered immune profiles as well as altered alveolar bone loss were all attributable to smoke exposure and/or F. alocis infection in vivo. Conclusions: F. alocis is well‐adapted to tobacco‐rich conditions and its pathogenesis is enhanced by tobacco smoke exposure. A smoke‐exposed ligature model of periodontitis shows promise as a tool with which to further unravel mechanisms underlying tobacco‐enhanced, bacteria‐induced disease. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a major oral pathogen that contributes to the development of periodontal disease. There is a significant degree of genetic variation among strains of P. gingivalis, and the population structure has been predicted to be panmictic, indicating that horizontal DNA transfer and recombination between strains are likely. The molecular events underlying this genetic exchange are not understood, although a putative type IV secretion system is present in the genome sequence of strain W83, implying that DNA conjugation may be responsible for genetic transfer in these bacteria. In this study, we provide in vitro evidence for the horizontal transfer of DNA using plasmid- and chromosome-based assays. In the plasmid assays, Bacteroides-derived shuttle vectors were tested for transfer from P. gingivalis strains into Escherichia coli. Of the eight strains tested, five were able to transfer DNA into E. coil by a mechanism most consistent with conjugation. Additionally, strains W83 and 33277 tested positive for the transfer of chromosomally integrated antibiotic resistance markers. Ten chimeras resulting from the chromosomal transfer assay were further analyzed by Southern hybridization and were shown to have exchanged DNA fragments of between 1.1 and 5.6 kb, but the overall strain identity remained intact. Chimeras showed phenotypic changes in the ability to accrete into biofilms, implying that DNA transfer events are sufficient to generate measurable changes in complex behaviors. This ability to transfer chromosomal DNA between strains may be an adaptation mechanism in the complex environment of the host oral cavity.

Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they are more susceptible to destructive periodontal disease and smokers may, ultimately, benefit from tobacco-specific preventive and treatment strategies. A Mariner transposon insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose products play roles in protein transport and catabolism, nicotinamide processing, protection against oxidative stress, drug resistance, and transcriptional regulation have all been identified as essential for CSE survival. Many of these tobacco-essential genes are also requisite for epithelial colonization and abscess formation, suggestive of a core stress-related P. gingivalis genome. Single-gene deletions in several of the TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon competition with the parent strain, under conditions of cigarette smoke extract-induced stress (1,000 ng/ml nicotine equivalents). This study identifies, for the first time, a subset of P. gingivalis genes required for surviving the plethora of insults present in cigarette smoke. Such conditionally essential genes may delineate bacterial persistence strategies and represent novel therapeutic foci for the prevention of P. gingivalis infection and related diseases in smokers and in general. [ABSTRACT FROM AUTHOR]

AT1 receptor subtype a (AT1Ra) expression is increased in the nucleus of the solitary tract (NTS) in spontaneously hypertensive rat (SHR) compared with Wistar Kyoto controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in the neural regulation of the peripheral inflammatory status and linked with hypertension. Transduction of brain neuronal cultures with recombinant adeno-associated virus type 2 (AAV2)-AT1R-small hairpin RNA (shRNA) resulted in a 72% decrease in AT1Ra mRNA and attenuated angiotensin II-induced increase in extracellular signal-regulated kinase 1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ≈30 mm Hg increase in the mean arterial pressure compared with control vector-injected animals (Sc-shRNA: 154±4 mm Hg; AT1R-shRNA: 183±10 mm Hg) and induced a resetting of the baroreflex control of heart rate to higher mean arterial pressure. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (CD90+, CD4- / CD5- / CD8-) and a 300% increase in the circulating inflammatory cells, including CD4+ + CD8+, CD45+ / 3+ T lymphocytes, and macrophages (CD68+). As a result, the endothelial progenitor cell/inflammatory cells ratio was decreased by 8- to 15-fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of Wistar Kyoto rats had no effect on mean arterial pressure and inflammatory cells. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counterhypertensive mechanism involving inflammatory/angiogenic cells. [ABSTRACT FROM AUTHOR]

AT1 receptor subtype a (AT1Ra) expression is increased in the nucleus of the solitary tract (NTS) in spontaneously hypertensive rat (SHR) compared with Wistar Kyoto controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in the neural regulation of the peripheral inflammatory status and linked with hypertension. Transduction of brain neuronal cultures with recombinant adeno-associated virus type 2 (AAV2)-AT1R-small hairpin RNA (shRNA) resulted in a 72% decrease in AT1Ra mRNA and attenuated angiotensin II-induced increase in extracellular signal-regulated kinase 1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ≈30 mmHg increase in the mean arterial pressure compared with control vector-injected animals (Sc-shRNA: 154±4 mm Hg; AT1R-shRNA: 183±10 mm Hg) and induced a resetting of the baroreflex control of heart rate to higher mean arterial pressure. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (CD90+ CD4-/CD5-/CD8-) and a 300% increase in the circulating inflammatory cells, including CD4++CD8+ CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the endothelial progenitor cell/inflammatory cells ratio was decreased by 8- to 15-fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of Wistar Kyoto rats had no effect on mean arterial pressure and inflammatory cells. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counterhypertensive mechanism involving inflammatory/angiogenic cells. [ABSTRACT FROM AUTHOR]

The article discusses research on the hypothesis brain (pro) renin receptor (PRR) as physiologically involved in cardiovascular homeostasis and the renin-angiotensin system (RAS) regulation of the brain. The study focused on the PRR in the supraoptic nucleus (SON) in the brain with higher PRR and messenger ribonucleic acid (mRNA) levels of spontaneously hypertensive rats (SHR). The study showed the function and role of brain PRR in the nueral control of cardiovascular functions showing the presence of intrinsic functional brain RAS.

AT1R expression is increased in the nucleus of solitary tract (NTS) of many animal models of hypertension. However, the impact of its chronic regulation on cardiovascular control is not well understood. In this study, we investigated the hypothesis that NTS AT1R is involved in peripheral inflammatory status and hypertension-linked cardiovascular pathophysiology. AAV2- mediated expression of a shRNA targeted the AT1R in the NTS has been used to test the hypothesis. First,the efficacy of the AAV2-AR1R-shRNA was established with the use of neuronal cells in primary culture. Transduction of these cultures by AAV2-AT1R-shRNA resulted in a 72% decrease in AT1R mRNA compared with the AAV2- Sc-shRNA transduced control. This was accompanied by complete attenuation of AngII-induced increase in ERK1/2 phosphorylation and neuronal action potential frequency (control: 2.2±0.4 vs. AT1R-shRNA: 0.5±0.3 hz). We then microinjected AAV2-AT1R-shRNA or control vector, AAV2-Scrambled (Sc)-shRNA into the NTS of 8-week-old SHR and WKY rats. Nine weeks following microinjection, AAV2-AT1R-shRNA group of SHRs showed a 29 mmHg increase in mean arterial pressure (MAP) (control: 154.2±4.7; shRNA: 183.0±10.2 mmHg), insignificant effects on the MAP of WKY rats was observed. The increase in MAP in the SHR by AAV2-AT1R-shRNA was associated with a 42% decrease in baroreflex bradycardia (control: 0.57±0.02; shRNA: 0.33±0.06 ms/mmHg), profound cardiac hypertrophy, and significant increase in the plasma norepinephrine (control: 6.36±0.39, shRNA: 13.0±4.13 ng/mL). In addition, AAV2-AT1R-shRNA rats showed significant increases in water intake (control: 32.5±1.5; shRNA: 12.5±2.5 ml/16h) and urine output (control: 1.6 ±0.5; shRNA: 9.3±2.5 ml/16h). Furthermore, AAV2-AT1R-shRNA treated SHR exhibited 74% decrease in circulating endothelial progenitor cells (EPC, CD90+, CD4/5/8-), and ∼300% increases in inflammatory cells (IC) including CD4/8+, T lymphocytes (CD45/3+) and macrophages (CD68+) . As a result, EPC/IC ratio was decreased by 8∼15 fold in AT1R-shRNA treated SHR. These observations suggest that increased AT1R in the NTS of SHR may present a counter-hypertensive mechanism involving inflammatory/angiogenic cells. [ABSTRACT FROM AUTHOR]