Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.
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ABSTRACT:
SUBMITTER: Mahfuz I
PROVIDER: S-EPMC4222080 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
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Project description:Epigenome-wide association studies (EWAS) assessing the link between DNA methylation (DNAm) and phenotypes related to structural brain measures, cognitive function, and neurodegenerative diseases are becoming increasingly more popular. Due to the inaccessibility of brain tissue in humans, several studies use peripheral tissues such as blood, buccal swabs, and saliva as surrogates. To aid the functional interpretation of EWAS findings in such settings, there is a need to assess the correlation of DNAm variability across tissues in the same individuals. In this study, we performed a correlation analysis between DNAm data of a total of n = 120 matched post-mortem buccal and prefrontal cortex samples. We identified nearly 25,000 (3% of approximately 730,000) cytosine-phosphate-guanine (CpG) sites showing significant (false discovery rate q < 0.05) correlations between buccal and PFC samples. Correlated CpG sites showed a preponderance to being located in promoter regions and showed a significant enrichment of being determined by genetic factors, i.e. methylation quantitative trait loci (mQTL), based on buccal and dorsolateral prefrontal cortex mQTL databases. Our novel buccal-brain DNAm correlation map will provide a valuable resource for future EWAS using buccal samples for studying DNAm effects on phenotypes relating to the brain. All correlation results are made freely available to the public online.
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Project description:AIM:Glucocorticoids play a major role in regulating the stress response, and an imbalance of glucocorticoids has been implicated in stress-related disorders. Within mouse models, CpGs across the genome have been shown to be differentially methylated in response to glucocorticoid treatment, and using the Infinium 27K array, it was shown that humans given synthetic glucocorticoids had DNA methylation (DNAm) changes in blood. However, further investigation of the extent to which glucocorticoids affect DNAm across a larger proportion of the genome is needed. METHODS:Buccal samples were collected before and after synthetic glucocorticoid treatment in the context of a dental procedure. This included 30 tooth extraction surgery patients who received 10 mg of dexamethasone. Genome-wide DNAm was assessed with the Infinium HumanMethylationEPIC array. RESULTS:Five CpGs showed genome-wide significant DNAm changes that were >10%. These differentially methylated CpGs were in or nearest the following genes: ZNF438, KLHDC10, miR-544 or CRABP1, DPH5, and WDFY2. Using previously published datasets of human blood gene expression changes following dexamethasone exposure, a significant proportion of genes with false-discovery-rate-adjusted significant CpGs were also differentially expressed. A pathway analysis of the genes with false-discovery-rate-adjusted significant CpGs revealed significant enrichment of olfactory transduction, pentose and glucuronate interconversions, ascorbate and aldarate metabolism, and steroid hormone biosynthesis pathways. CONCLUSION:High-dose synthetic glucocorticoid administration in the setting of a dental procedure was significantly associated with DNAm changes within buccal samples. These findings are consistent with prior findings of an influence of glucocorticoids on DNAm in humans.
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Project description:BackgroundIn 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections.ResultsDNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 ?g of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay.ConclusionsMouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.
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Project description:Streptococcus parasanguinis overview
| PRJNA48543 | ENA
Project description:This study aimed to test the sterility of new unused orthodontic buccal tubes received from manufacturers. Four different types of buccal tubes were used straight from the manufactures package without any additional sterilizing step. Of these buccal tubes tested, three genera of bacteria, implicated as opportunistic pathogens, namely Micrococcus luteus, Staphylococcus haemolyticus and Acinetobacter calcoaceticus were recovered from these buccal tubes. Our data showing microbial contamination on buccal tubes highlights the need of sterilization before clinical use. We also suggest that manufacturers should list the sterility state of orthodontic buccal tubes on their packaging or instructions stating the need for sterilization.
| S-EPMC2956099 | biostudies-literature
Project description:DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies.
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Project description:Modern human contamination is a common problem in ancient DNA studies. We provide evidence that this issue is also present in studies in great apes, which are our closest living relatives, for example in noninvasive samples. Here, we present a simple method to detect human contamination in short-read sequencing data from different species: HuConTest. We demonstrate its feasibility using blood and tissue samples from these species. This test is particularly useful for more complex samples (such as museum and noninvasive samples) which have smaller amounts of endogenous DNA, as we show here.
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Project description:Dental biofilm formation is critical for maintaining the healthy microbial ecology of the oral cavity. Streptococci are predominant bacterial species in the oral cavity and play important roles in the initiation of plaque formation. In this study, we identified a new cell surface protein, BapA1, from Streptococcus parasanguinis FW213 and determined that BapA1 is critical for biofilm formation. Sequence analysis revealed that BapA1 possesses a typical cell wall-sorting signal for cell surface-anchored proteins from Gram-positive bacteria. No functional orthologue was reported in other streptococci. BapA1 possesses nine putative pilin isopeptide linker domains which are crucial for pilus assembly in a number of Gram-positive bacteria. Deletion of the 3' portion of bapA1 generated a mutant that lacks surface-anchored BapA1 and abolishes formation of short fibrils on the cell surface. The mutant failed to form biofilms and exhibited reduced adherence to an in vitro tooth model. The BapA1 deficiency also inhibited bacterial autoaggregation. The N-terminal muramidase-released-protein-like domain mediated BapA1-BapA1 interactions, suggesting that BapA1-mediated cell-cell interactions are important for bacterial autoaggregation and biofilm formation. Furthermore, the BapA1-mediated bacterial adhesion and biofilm formation are independent of a fimbria-associated serine-rich repeat adhesin, Fap1, demonstrating that BapA1 is a new streptococcal adhesin.
| S-EPMC3147580 | biostudies-literature
Project description:Streptococcus parasanguinis is a dominant isolate of dental plaque and an opportunistic pathogen associated with subacute endocarditis. As the expression of collagen binding proteins (CBPs) could promote the establishment of S. parasanguinis in the host, the functions of three putative CBP-encoding loci, Spaf_0420, Spaf_1570, and Spaf_1573, were analyzed using isogenic mutant strains. It was revealed that S. parasanguinis FW213 bound effectively to fibronectin and type I collagen, but the strain's affinity for laminin and type IV collagen was quite low. By using various deletion derivatives, it was found that these three loci mediated the binding of S. parasanguinis to multiple extracellular matrix molecules, with type I collagen as the common substrate. Derivative strains with a deletion in any of the three loci expressed reduced binding to trypsin-treated swine heart valves. The deletion of these loci also reduced the viable count of S. parasanguinis bacteria within macrophages, especially the loss of Spaf_0420, but only strains with deletions in Spaf_0420 and Spaf_1570 expressed reduced virulence in the Galleria mellonella larva model. The deletion of Spaf_1570 and Spaf_1573 affected mainly the structure, but not the overall mass, of biofilm cultures in a flow cell system. Thus, CBPs are likely to be more critical for the initial colonization of S. parasanguinis on host tissues during the development of endocarditis.IMPORTANCE Bacteria generally can utilize multiple adhesins to establish themselves in the host. We found that Streptococcus parasanguinis, a dominant oral commensal and an opportunistic pathogen for subacute endocarditis, possesses at least three collagen-binding proteins that enable S. parasanguinis to successfully colonize damaged heart tissues and escape innate immune clearance. The binding specificities of these three proteins for extracellular matrix molecules differ, although all three proteins participate in biofilm formation by S. parasanguinis The "multiligand for multisubstrate" feature of these adhesins may explain the high adaptability of this microbe to different tissue sites.
| S-EPMC7565896 | biostudies-literature
Project description:BackgroundStreptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation.ResultsDeletion and site-directed mutagenesis were employed to dissect the regions within Gap3 that were important for its function in Fap1 glycosylation and biogenesis. A deletion of 6 consecutive amino acids, PDLPIL, eliminated the production of the mature 200 kDa Fap1 protein and gave rise instead to a 470 kDa Fap1 intermediate that was only partially glycosylated. Site-directed mutagenesis of the 6 amino acids revealed that only three of these amino acids were required. Mutants in these amino acids (L64R, P65R and L67T) produced the premature 470 kDa Fap1 intermediate. Mutants in the remaining amino acids produced the mature form of Fap1. Cell surface expression of the Fap1 precursor among L64R, P65R and L67T mutants was reduced to levels consistent with that of a gap3 insertional mutant. Electron micrographs showed that these 3 mutants lost their long peritrichous fimbriae. Furthermore, their in vitro adhesion ability to saliva-coated hydroxylapatite (SHA) was inhibited.ConclusionOur data suggest that 3 highly conserved, hydrophobic residues L64, P65 and L67 in Gap3 are essential for Gap3 function and are important for complete glycosylation of Fap1, fimbrial formation and bacterial adhesion.
| S-EPMC2335109 | biostudies-literature