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Cerda-Costa N.,Institute of Barcelona | Guevara T.,Institute of Barcelona | Karim A.Y.,Jagiellonian University | Ksiazek M.,Jagiellonian University | And 6 more authors.
Molecular Microbiology | Year: 2011

Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52 kDa multidomain protein comprising a central 18 kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analysed the 3D structure of Kly18 in the absence and presence of Mg2+ or Ca2+, which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side-chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg2+ or Ca2+ binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally characterized non-mammalian MMP is a xenologue co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs. © 2010 Blackwell Publishing Ltd.

Nadkarni M.A.,Institute of Dental Research | Simonian M.R.,Institute of Dental Research | Harty D.W.S.,Institute of Dental Research | Zoellner H.,University of Sydney | And 2 more authors.
Journal of Clinical Microbiology | Year: 2010

In earlier studies we used molecular methods to identify the major bacterial consortia associated with advanced dentin caries. These consortia are dominated by bacteria from the families Lactobacillaceae, Streptococcaceae, Veillonellaceae (formerly Acidaminococcaceae), Eubacteriaceae, and Lachnospiraceae from the phylum Firmicutes; Coriobacteriaceae, Bifidobacteriaceae, and Propionibacteriaceae from the phylum Actinobacteria; and Prevotellaceae from the phylum Bacteroidetes, as well as fusobacteria. The phases of infection of vital pulp tissue by dentin microorganisms remain obscure. In the present study, fluorescence in situ hybridization was performed on sections of tissue embedded in resin. Probes for 16S rRNA corresponding to the major taxa of bacteria in carious dentin were used to provide information on the characteristics of pulp infection. Lactobacilli were prominent in 7 of 8 pulps determined to be at a limited stage of infection. Established infection (6 pulps) showed a more complex profile, with lactobacilli persisting in all of the lesions and with invasion of the necrotic regions of tissue by Bacteroidetes, fusobacteria, Lachnospiraceae, and Coriobacteriaceae in particular. Advanced infections (7 pulps) were characterized by mixed anaerobic species, with a strong representation by Coriobacteriaceae and Lachnospiraceae. Lactobacilli were not represented at this stage. Typically, groups of organisms were spatially isolated within the pulp tissue. Analysis indicated that lactobacilli could invade vital pulp tissue to achieve a very high biomass that was not associated with a detectable local inflammatory infiltrate. The findings establish that invasion of the dental pulp can be associated with a pronounced selection from the complex microbial populations within carious dentin, suggesting specific pathogenicity. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

News Article
Site: www.biosciencetechnology.com

A brain region controlling whether we feel happy or sad, as well as addiction, is remodeled by chronic pain, reports a new Northwestern Medicine study. And in a significant breakthrough for the millions of Americans suffering from chronic pain, scientists have developed a new treatment strategy that restores this region and dramatically lessens pain symptoms in an animal model. The new treatment combines two FDA-approved drugs: a Parkinson’s drug, L-dopa, and a non-steroidal anti-inflammatory drug. The combined drugs target brain circuits in the nucleus accumbens and completely eliminate chronic pain behavior when administered to rodents with chronic pain. The key is administering the drugs together and shortly after an injury. As a result of the study’s findings, the scientists are pursuing a clinical trial. The treatment has the potential to prevent chronic pain if used early enough after injury, the scientists said. The study was published December 21 in Nature Neuroscience. ‘It was surprising to us that chronic pain actually rewires the part of the brain controlling whether you feel happy or sad,” said corresponding author D. James Surmeier, chair of physiology at Northwestern University Feinberg School of Medicine. “By understanding what was causing these changes, we were able to design a corrective therapy that worked remarkably well in the models. The question now is whether it will work in humans.” “The study shows you can think of chronic pain as the brain getting addicted to pain,” said A. Vania Apkarian, also a corresponding author and a professor of physiology at Feinberg. “The brain circuit that has to do with addiction has gotten involved in the pain process itself.” A group of neurons thought to be responsible for negative emotions became hyper-excitable and more strongly connected with other regions of the brain linked to feeling bad within days after an injury that triggers chronic pain behavior, the study showed. It went on to show this change was triggered by a drop in dopamine, a critical neurotransmitter. When scientists administered the non-steroidal anti-inflammatory drug and L-dopa, which raises dopamine levels, the changes in the brain were reversed and the animals’ chronic pain behavior stopped. “These results establish chronic pain cannot be viewed as a purely sensory phenomenon but instead is closely related to emotions,” Apkarian said. In addition, Northwestern scientists treated rats experiencing chronic pain with another Parkinson’s drug, pramipexole, that activated dopamine receptors, mimicking dopamine’s effect. This drug also decreased the animals’ pain-like behavior. “It is remarkable that by changing the activity of a single cell type in an emotional area of the brain, we can prevent the pain behavior,” said Marco Martina, associate professor of physiology at Feinberg and also a corresponding author. Currently, the most common treatment for chronic pain is a non-steroidal anti-inflammatory type of drug, which has limited effectiveness. “The treatments for chronic pain we currently have are very limited,” said Surmeier, also the Nathan Smith Davis Professor of Physiology. The results of the study suggest supplementing anti-inflammatories with a medication that activates dopamine receptors or raises dopamine levels might be more effective in treating chronic pain and/or preventing a transition to chronic pain. Chronic pain is an intractable problem for millions of Americans. It’s the number one cause of disability in the U.S. and costs more than $600 billion per year in in health care. An estimated 20 percent of the U.S. and world population suffers from chronic pain, reports the World Health Organization and the National Academy of Sciences. This work was supported by grant DE022746 from the National Institute of Dental Research and grants from National Institute of Neurological Disorders and Stroke, of the National Institutes of Health.

Eaton R.E.,Institute of Dental Research | Jacques N.A.,Institute of Dental Research
Molecular Oral Microbiology | Year: 2010

Previous studies identified nine genes with increased expression in Streptococcus mutans biofilms of which six possessed putative ComX promoter sequences and were homologous to competence-induced genes in Streptococcus pneumoniae, Streptococcus gordonii and Bacillus subtilis. As competence increases in biofilms, a study was undertaken into the roles that these biofilm-induced genes might play in transformation. Only five of the nine gene deletions had a significant effect on transformation efficiency. Deletion of the genes for recombinase A, recA, DNA processing protein, dprA and single-stranded DNA-binding protein, ssbA, produced results comparable with those from other bacteria, supporting the contention that these proteins have similar functions in S. mutans competence. The uncharacterized genes SMU.769 and SMU.836 produced results in variance to deletion mutants of putative homologues in S. pneumoniae. Deletion of SMU.769 reduced chromosomal transformation 2.3-fold. SMU.769 belongs to a family of conserved genes induced by the competence-stimulating peptide and which have no established function. In contrast, deletion of SMU.836 reduced transformation of both plasmid and chromosomal DNA to <3%. Homology searches suggested that Smu.836 belongs to a family of competence-induced peptidoglycan hydrolases with a conserved enzyme domain and a species-variable cell-binding domain for which the best characterized member is the choline-binding protein D, CbpD, of S. pneumoniae. © 2010 John Wiley & Sons A/S.

Nadkarni M.A.,Institute of Dental Research | Nadkarni M.A.,University of Sydney | Chen Z.,University of New South Wales | Wilkins M.R.,University of New South Wales | And 2 more authors.
PLoS ONE | Year: 2014

The human oral microbiome has a major role in oral diseases including dental caries. Our studies on progression of caries infection through dentin and more recently, the invasion of vital dental pulp, detected Lactobacillus rhamnosus in the initial stages of infection of vital pulp tissue. In this study employing current high-throughput next generation sequencing technology we sought to obtain insight into genomic traits of tissue invasive L. rhamnosus, to recognise biomarkers that could provide an understanding of pathogenic potential of lactobacilli, generally regarded as safe. Roche GS FLX+ technology was used to generate whole genome sequences of two clinical isolates of L. rhamnosus infecting vital pulp. Detailed genome-wide comparison of the genetic profiles of tissue invasive L. rhamnosus with probiotic L. rhamnosus was performed to test the hypothesis that specific strains of L. rhamnosus possessing a unique gene complement are selected for the capacity to invade vital pulp tissue. Analysis identified 264 and 258 genes respectively, from dental pulp-invasive L. rhamnosus strains LRHMDP2 and LRHMDP3 isolated from two different subjects that were not present in the reference probiotic L. rhamnosus strain ATCC 53103 (GG). Distinct genome signatures identified included the presence of a modified exopolysaccharide cluster, a characteristic confirmed in a further six clinical isolates. Additional features of LRHMDP2 and LRHMDP3 were altered transcriptional regulators from RpoN, NtrC, MutR, ArsR and zinc-binding Cro/CI families, as well as changes in the two-component sensor kinase response regulator and ABC transporters for ferric iron. Both clinical isolates of L. rhamnosus contained a single SpaFED cluster, as in L. rhamnosus Lc705, instead of the two Spa clusters (SpaCBA and SpaFED) identified in L. rhamnosus ATCC 53103 (GG). Genomic distance analysis and SNP divergence confirmed a close relationship of the clinical isolates but segregation from the reference probiotic L. rhamnosus strain ATCC 53103 (GG). © 2014 Nadkarni et al.

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