Project description:To look for transcriptomic changes that resulted in membrane vesicle biogenesis, we performed RNA sequencing on S.sanguinis in different culture and community conditions We then examined three separate differential expression analyses, as well as correlated the phenotypic vesicle quantification with normalized count data across all samples.
Project description:Streptococcus sanguinis, a Gram-positive oral commensal, is highly prevalent in the oral cavity and well-characterized for its ability to antagonize oral pathobionts. We have found that production and dissemination of membrane vesicles by S. sanguinis is dependent on environmental and community factors. Co-culture with interacting commensal Corynnyebacterium durum, as well as with the periodontal pathobiont Filifactor alocis had no effect on S. sanguinis vesicle number and size, whereas the periodontal pathobiont Porphyromonas gingivalis abolished S. sanguinis vesicle production. Using both correlation and differential expression analyses to examine the transcriptomic changes underlying vesicle production, we found that differential expression of genes encoding proteins related to the cytoplasmic membrane and peptidoglycan correlate with the abundance of membrane vesicles. Proteomic characterizations of the vesicle cargo identified a variety of proteins, including those predicted to influence host interactions or host immune responses. Cell culture studies of gingival epithelial cells demonstrated that both crude and highly purified membrane vesicles could induce the expression of IL-8, TNF-α, IL-1β, and Gro-α within 6 hours of inoculation at levels comparable to whole cells. OTaken together, our findings suggest that production of membrane vesicles by S. sanguinis is heavily influenced by community and environmental factors, and plays an important role in communication with host cells.
Project description:Mycobacterium tuberculosis (Mtb) secretes pathogenicity factors and immunologically active molecules via membrane vesicles. However, nothing is known about the mechanisms involved in mycobacterial vesicle biogenesis. This study investigates molecular determinants of membrane vesicle production in Mtb by analyzing Mtb cells under conditions of high vesicle production: iron limitation and VirR restriction. Ultrastructural analysis showed extensive cell envelope restructuring in association with vesicle release that correlated with downregulation of cell surface lipid biosynthesis and peptidoglycan alterations. Comparative transcriptomics showed common upregulation of the iniBAC operon in association with high vesicle production in Mtb cells. Vesicle production analysis demonstrated that the dynamin-like proteins (DLPs) encoded by this operon, IniA and IniC, are necessary for release of EV by Mtb in culture and in infected macrophages. Isoniazid, a first-line antibiotic, used in tuberculosis treatment, was found to stimulate vesicle release in a DLP-dependent manner. Our results provide a new understanding of the function of mycobacterial DLPs and mechanistic insights into vesicle biogenesis. The findings will enable further understanding of the relevance of Mtb-derived extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for therapeutic research.
Project description:Transcriptional profiling of ssa_1972-null mutant of Streptococcus sanguinis compared with wild type. The ssa_1972 gene was inactivated in Streptococcus sanguinis SK36 and transcriptional profile was compared with wild type SK36. More information can be found at http://www.people.vcu.edu/~pingxu One-condition experiment, M-NM-^Tssa_1972 vs S. sanguinis SK36 cells. Biological replicates: 3 wild type, 3 M-NM-^Tssa_1972, independently grown and harvested. One replicate (one wild type and one M-NM-^Tssa_1972 mixture) per array.
Project description:Transcriptional profiling of ssa_1972-null mutant of Streptococcus sanguinis compared with wild type. The ssa_1972 gene was inactivated in Streptococcus sanguinis SK36 and transcriptional profile was compared with wild type SK36. More information can be found at http://www.people.vcu.edu/~pingxu
Project description:Streptococcus sanguinis is a major component of the oral flora and an important cause of infective endocarditis. The genome sequence of S. sanguinis strain SK36 was recently determined. A number of foreign genes acquired by natural transformation were detected, as well as orthologs of competence genes previously identified in other species. However, significant differences in the S. sanguinis competence system relative to that of other streptococci were noted. We sought to examine S. sanguinis genetic competence, to characterize the global transcriptional response to competence induction, and to compare our results with those obtained from previous analyses of other streptococci. A mutant possessing an in-frame deletion in the comC gene encoding the competence-stimulating peptide was created and confirmed to have the expected phenotype. Studies indicated that competence could be induced in this strain by addition of competence-stimulating peptide, and determined the optimal conditions to employ for this purpose. Expression was monitored by microarray analysis at multiple time points from 2.5 to 30 min after induction. Over 200 genes were identified whose expression was altered at least two-fold in at least one time point, with the majority upregulated. The M-bM-^@M-^\lateM-bM-^@M-^] response was typical of that seen in previous studies. However, comparison of the M-bM-^@M-^\earlyM-bM-^@M-^] response in S. sanguinis with that of other streptococci revealed unexpected heterogeneity with regard to the number of genes induced, the nature of these genes, and their putative upstream regulatory sequences. S. sanguinis possesses a comparatively limited early response, which may define a minimal competence regulatory circuit. Transcriptional analysis of S. sanguinis strain JFP41 cells 0 to 30 min after treatment with CSP. Biological replicates: 3 replicates each independently grown and harvested. 4 technical replicates per array
Project description:Streptococcus sanguinis is a major component of the oral flora and an important cause of infective endocarditis. The genome sequence of S. sanguinis strain SK36 was recently determined. A number of foreign genes acquired by natural transformation were detected, as well as orthologs of competence genes previously identified in other species. However, significant differences in the S. sanguinis competence system relative to that of other streptococci were noted. We sought to examine S. sanguinis genetic competence, to characterize the global transcriptional response to competence induction, and to compare our results with those obtained from previous analyses of other streptococci. A mutant possessing an in-frame deletion in the comC gene encoding the competence-stimulating peptide was created and confirmed to have the expected phenotype. Studies indicated that competence could be induced in this strain by addition of competence-stimulating peptide, and determined the optimal conditions to employ for this purpose. Expression was monitored by microarray analysis at multiple time points from 2.5 to 30 min after induction. Over 200 genes were identified whose expression was altered at least two-fold in at least one time point, with the majority upregulated. The “late” response was typical of that seen in previous studies. However, comparison of the “early” response in S. sanguinis with that of other streptococci revealed unexpected heterogeneity with regard to the number of genes induced, the nature of these genes, and their putative upstream regulatory sequences. S. sanguinis possesses a comparatively limited early response, which may define a minimal competence regulatory circuit.