मौखिक Biofilms की व्यापक बायोकेमिकल, स्ट्रक्चरल और transcriptome मूल्यांकन के लिए एक विश्लेषणात्मक उपकरण बॉक्स अपरिवर्तक Streptococci द्वारा मध्यस्थता
Summary
दांत सतहों पर गठित Biofilms अत्यधिक जटिल और लगातार सहज और exogenous पर्यावरणीय चुनौतियों, जो अपनी वास्तुकला मिलाना फिजियोलॉजी और transcriptome को उजागर कर रहे हैं. हम एक Toolbox विकसित करने के लिए संरचना, संरचनात्मक संगठन और मौखिक biofilms के जीन की अभिव्यक्ति, जो biofilm अनुसंधान के अन्य क्षेत्रों के लिए अनुकूलित किया जा सकता है की जांच.
Abstract
Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.
Protocol
Discussion
इस प्रस्तुति में, हम विश्लेषणात्मक उपकरण बॉक्स (ईपीएस / बैक्टीरिया इमेजिंग और माइक्रोएरे डेटा / खनन प्रसंस्करण), और विभिन्न प्रणाली में एकीकृत assays के बहुमुखी प्रतिभा और उपयोगिता के दो महत्वपूर्ण घटक का …
Divulgations
The authors have nothing to disclose.
Acknowledgements
लेखकों MDV के विकास के लिए डॉ. गैरी Xie और हरबर्ट ली धन्यवाद देना चाहूंगा. हम भी डीआरएस धन्यवाद. सिमोन ड्यूआर्टे, रेमिरो Murata, जॅ-Gyu Jeon, जैकलिन Abranches, और अपने उपकरण बॉक्स के विश्लेषणात्मक घटकों के लिए तकनीकी और वैज्ञानिक योगदान के लिए सुश्री स्टेसी ग्रेगोइरे. इस अध्ययन USPHS अनुसंधान अनुदान DE018023 द्वारा दंत चिकित्सा और craniofacial रिसर्च राष्ट्रीय संस्थान से हिस्से में समर्थित किया गया था.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Syto 9 | Invitrogen | S34854 | ||
| Syto 60 | Invitrogen | S11342 | ||
| Dextran conjugated alexa 647 | Invitrogen | D22914 | ||
| Olympus FV1000 two-photon laser scanning microscope | Olympus, Tokyo, Japan |
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