Studies on the stress response in Fusobacterium nucleatum.

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism belonging to the so-called ‘orange complex’ which is believed to play an important role in the microbial succession associated with the pathogenesis of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the progression from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. Studies have shown that F. nucleatum is partly responsible for the rise in pH and have concluded that in comparison to other plaque inhabitants, F. nucleatum has the greatest ability to neutralise acidic environments. In common with a number of other oral bacteria, F. nucleatum has also been shown to produce intracellular polyglucose (IP) from simple sugars such as glucose, galactose and fructose. Its response and adaptation to stressful environmental conditions such as pH is unknown. The overall aim of this study was, therefore, to determine how F. nucleatum copes with environmental stresses induced by pH changes. F. nucleatum was grown by continuous culture in a chemically defined medium at a growth rate corresponding to those measured in vivo. The effect on protein expression, and IP synthesis was examined during steady-state growth at high (>7.2<7.8) or low pH (pH 6.4). The present study also investigated the response of F. nucleatum to growth at pH 8.2. It was found that the organism grew as a biofilm and this corresponded with an increase in cellular hydrophobicity and decreased IP levels. Optimal growth pH’s differed between the different sub-species used in this study. In response to pH stress, F. nucleatum changed its amino acid and glucose utilisation and increased IP synthesis at the expense of cell numbers. Pulsing the chemostat with glutamic acid or serine produced an increase in IP synthesis and the pattern of end-products observed was dependent upon the amino acid being fermented. The effect on IP synthesis in response to increased levels of exogenous fermentable amino acids was also compared during concomitant fructose or glucose fermentation. Growth media containing fermentable amino acids and supplemented with fructose produced higher cell numbers and non-detectable levels of IP compared to media containing glucose. The differential expression of cytoplasmic- and cell envelope-proteins induced by changes in pH were identified by two-dimensional gel electrophoresis. The results represent the first proteomic investigation of F. nucleatum. Twenty-two cytoplasmic proteins were found to have altered expression in response to external pH. At low (sub-optimal) pH, proteins associated with the generation of ATP and ammonia were up-regulated, the latter contributing to the alkalinisation of the gingival sulcus. Conversely, neutral to alkaline pH conditions led to the upregulation of enzymes involved in energy storage. The study also identified several proteins associated with iron limitation and fatty acid synthesis which might not otherwise have been identified as part of the pH-dependent response. In response to growth at pH 7.8, 14 cell envelope proteins were identified as having significantly altered expression. Down-regulated proteins included those associated with uptake of C4 di-carboxylates and phosphorus, a potential membrane protease and an enzyme associated with amino acid fermentation. The up-regulation of a transcriptional regulator linked to the repression of sugar metabolism was also reported along with proteins linked to the transport of iron. The periplasmic chaperone, peptidyl prolyl cis trans isomerase, which is responsible for the folding of outer membrane proteins, was also found to be up-regulated. In conclusion, the proteomic investigation of protein expression by F. nucleatum identified gene products which form part of the organism’s coordinated stress response to changes in environmental pH. In addition to these, the physiological based studies also presented help to explain the organism’s persistence during the transition from health to disease in vivo.Thesis (Ph.D.) - University of Adelaide, Dental School, 200

The response to oxidative stress ofFusobacterium nucleatumgrown in continuous culture

TesisEl presente trabajo de investigación tuvo como problema ¿Cuál es la calidad de las sentencias de primera y segunda instancia sobre fijación de pensión alimenticia según los parámetros normativos, doctrinarios y jurisprudenciales pertinentes, en el expediente N° 01132- 2013-0-2501-JP-FC-02, del Distrito Judicial del Santa – 2018?; El objetivo fue: determinar la calidad de las sentencias en estudio. Es de tipo, cuantitativo cualitativo, nivel exploratorio descriptivo, y diseño no experimental, retrospectivo y transversal. La unidad de análisis fue un expediente judicial seleccionado mediante muestreo por conveniencia, para recolectar los datos se utilizaron las técnicas de la observación y el análisis de contenido, y como instrumento una lista de cotejo validada mediante juicio de expertos. Los resultados revelaron que la calidad de la parte expositiva, considerativa y resolutiva, pertenecientes a: la sentencia de primera instancia fue de rango: alta, muy alta y muy alta; y de la sentencia de segunda instancia: muy alta, muy alta y alta. Se concluyó, que la calidad de ambas sentencias, fueron de rango muy alta, respectivamente

The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis

Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are bacterial species which frequently co-colonise the nasopharynx, but can also transit to the middle ear to cause otitis media. Chronic otitis media is often associated with a polymicrobial infection by these bacteria. However, despite being present in polymicrobial infections, the molecular interactions between these bacterial species remain poorly understood. We have previously reported competitive interactions driven by pH and growth phase between H. influenzae and S. pneumoniae. In this study, we have revealed competitive interactions between the three otopathogens, which resulted in reduction of H. influenzae viability in co-culture with S. pneumoniae and in triple-species culture. Transcriptomic analysis by mRNA sequencing identified a central role of arginine in mediating these interactions. Arginine supplementation was able to increase H. influenzae survival in a dual-species environment with S. pneumoniae, and in a triple-species environment. Arginine was used by H. influenzae for ATP production, and levels of ATP generated in dual- and triple-species co-culture at early stages of growth were significantly higher than the combined ATP levels of single-species cultures. These results indicate a central role for arginine-mediated ATP production by H. influenzae in the polymicrobial community

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population

Subpopulations in Strains of <i>Staphylococcus aureus</i> Provide Antibiotic Tolerance

The ability of Staphylococcus aureus to colonise different niches across the human body is linked to an adaptable metabolic capability, as well as its ability to persist within specific tissues despite adverse conditions. In many cases, as S. aureus proliferates within an anatomical niche, there is an associated pathology. The immune response, together with medical interventions such as antibiotics, often removes the S. aureus cells that are causing this disease. However, a common issue in S. aureus infections is a relapse of disease. Within infected tissue, S. aureus exists as a population of cells, and it adopts a diversity of cell types. In evolutionary biology, the concept of “bet-hedging” has established that even in positive conditions, there are members that arise within a population that would be present as non-beneficial, but if those conditions change, these traits could allow survival. For S. aureus, some of these cells within an infection have a reduced fitness, are not rapidly proliferating or are the cause of an active host response and disease, but these do remain even after the disease seems to have been cleared. This is true for persistence against immune responses but also as a continual presence in spite of antibiotic treatment. We propose that the constant arousal of suboptimal populations at any timepoint is a key strategy for S. aureus long-term infection and survival. Thus, understanding the molecular basis for this feature could be instrumental to combat persistent infections

D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment.

Enterococcus faecalis is the most frequent species present in post-treatment disease and plays a significant role in persistent periapical infections following root canal treatment. Its ability to persist in stressful environments is inter alia, due to its ability to form biofilms. The presence of certain D-amino acids (DAAs) has previously been shown to reduce formation of Bacillus subtilis biofilms. The aims of this investigation were to determine if DAAs disrupt biofilms in early and late growth stages for clinical E. faecalis strains and to test their efficacy in disrupting E. faecalis biofilms grown in sub-minimum inhibitory concentrations of commonly used endodontic biocides. From thirty-seven E. faecalis strains, the ten "best" biofilm producers were used to test the ability of a mixture containing D-leucine, D-methionine, D-tyrosine and D-tryptophan to reduce biofilm growth over a period of 24, 72 and 144 hours and when compared to their cognate L-Amino Acids (LAAs). We have previously shown that sub-MIC levels of tetracycline and sodium hypochlorite promotes biofilm growth in clinical strains of E. faecalis. DAAs were therefore tested for their effectiveness to reduce biofilm growth in the presence of sub-minimal concentrations of sodium hypochlorite (NaOCl-0.031%) and Odontocide™ (0.25% w/v), and in the presence of Odontopaste™ (0.25% w/v). DAAs significantly reduced biofilm formation for all strains tested in vitro, while DAAs significantly reduced biofilm formation compared to LAAs. The inhibitory effect of DAAs on biofilm formation was concentration dependent. DAAs were also shown to be effective in reducing E. faecalis biofilms in the presence of Odontopaste™ and sub-MIC levels of NaOCl and Odontocide™. The results suggest that the inclusion of DAAs into current endodontic procedures may reduce E. faecalis biofilms

Polycationic Silver Nanoclusters Comprising Nanoreservoirs of Ag+ Ions with High Antimicrobial and Antibiofilm Activity

Silver-based nano-antibiotics are rapidly developing as promising alternatives to conventional antibiotics. Ideally, to remain potent against a wide range of drug-resistant and anaerobic bacteria, silver-based nano-antibiotics should easily penetrate through the bacterial cell walls and actively release silver ions. In this study, highly monodispersed, ultrasmall (50%) of silver ions (i.e., Ag+ nanoreservoirs) on the cluster surface maintains their efficiency in both aerobic and anaerobic conditions. Significantly, the pAgNCs showed a strong capacity to significantly delay the development of bacterial resistance when compared to similar-sized negatively charged silver nanoparticles or conventional antibiotics. This study demonstrates a novel design strategy that can lay the foundation for the development of future highly potent nano-antibiotics effective against a broad spectrum of pathogens and biofilms needed in many everyday life applications and industries

Abnormal pregnancy outcomes in mice using an induced periodontitis model and the haematogenous migration of Fusobacterium nucleatum sub-species to the murine placenta.

To investigate if there is subspecies specific migration to the placenta by Fusobacterium nucleatum (Fn) and to determine whether experimentally induced periodontitis results in adverse pregnancy outcomes (APO) in mice.Periodontitis was induced in pregnant mice using an inoculum of Fn and Porphyromonas gingivalis. In parallel, four sub-species of Fn were individually injected into the circulatory system. At day 18 of gestation, the placenta, liver, spleen and blood were harvested and litter size, number of viable fetuses and resorptions, maternal, fetal and placenta weights were recorded. For the direct inoculation group, some mice were allowed to deliver for assessment of length of gestation, litter size, maternal, placental and pup weight. The presence of Fn was assessed by PCR and inflammatory mediators were measured by ELISA or multiplex analysis.Mice with alveolar bone loss, a marker of periodontitis, demonstrated significantly higher fetal weights (p = 0.015) and fetal/placental weight ratios (p = 0.030). PCR analysis of maternal organs did not identify Fn in any extracted tissues. In mice that received direct injection of Fn subspecies, varying degrees of APO were observed including preterm birth, intrauterine growth restriction, and fetal loss. Haematogenous spread of only Fn subsp. nucleatum to the placenta was confirmed. Litter size was significantly smaller (p = 0.023) and the number of resorptions was higher in inoculated versus control groups. Mice injected with subsp. nucleatum had significantly increased circulating CRP levels (p = 0.020) compared to controls while the mice with induced periodontitis had increased levels of IL-6 (p = 0.047) and IL-8 (p = 0.105).Periodontitis in mice elevated fetal weight and the fetal weight/placental weight ratio. This study found that subsp. nucleatum migrated haematogenously to the placenta, leading to APO in mice. The study supports the potential role of Fn in the association between periodontitis and APO

Probiotic Lactobacillus Rhamnosus GG Protects Against P. Gingivalis And F. Nucleatum Gut Dysbiosis.

OBJECTIVES: This study investigated changes induced by Porphyromonas gingivalis and on gastrointestinal histology and gut microbiome in a mouse model of experimental periodontitis. The effect of probiotic Lactobacillus rhamnosus GG (LGG) in altering these changes was also investigated. METHODS: IThirty-six mice were allocated into six groups. Experimental alveolar bone loss was induced by oral inoculation with P. gingivalis and F. nucleatum. LGG was orally inoculated or orally gavaged. Gastrointestinal tissue changes were assessed using histological analysis and immunohistochemistry. Caecal microbiome was analysed by sequencing 16S rRNA genes of caecal content. RESULTS: Inoculation with P. gingivalis and F. nucleatum induced inflammation throughout gastrointestinal tract (p less than 0.05), increased expression of IL-6 in ileum (p = 0.052) and altered composition of caecal microbiome (p less than 0.05) in experimental mice compared to controls. Mice treated with LGG had reduced tissue inflammation in duodenum (p = 0.044) and lowered levels of IL-6 in ileum (p = 0.048) when compared with disease. LGG therapy influenced gut microbiome changes. CONCLUSION: P. gingivalis and F. nucleatum inoculation induced significant changes in intestinal inflammation and caecal microbiome. Oral gavage with LGG exerted a protective effect against intestinal inflammation and limited gut microbiome changes associated with P. gingivalis and F. nucleatum