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Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing.

ABSTRACT: Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force1,2. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cell reporter of this operon, which we name the flow-regulated operon (fro), we establish that P. aeruginosa dynamically tunes gene expression to flow intensity through a process we call rheosensing (as rheo- is Greek for flow). We further show that rheosensing occurs in multicellular biofilms, involves signalling through the alternative sigma factor FroR, and does not require known surface sensors. To directly test whether rheosensing measures force, we independently altered the two parameters that contribute to shear stress: shear rate and solution viscosity. Surprisingly, we discovered that rheosensing is sensitive to shear rate but not viscosity, indicating that rheosensing is a kinematic (force-independent) form of mechanosensing. Thus, our findings challenge the dominant belief that biological mechanosensing requires the measurement of forces.

SUBMITTER: Sanfilippo JE

PROVIDER: S-EPMC6656604 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing.

Sanfilippo Joseph E JE Lorestani Alexander A Koch Matthias D MD Bratton Benjamin P BP Siryaporn Albert A Stone Howard A HA Gitai Zemer Z

Nature microbiology 20190513 8

Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force<sup>1,2</sup>. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cel ...[more]

PMID: 31086313

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Project description:BackgroundMucosal infections elicit inflammatory responses via regulated signaling pathways. Infection outcome depends strongly on early events occurring immediately when bacteria start interacting with cells in the mucosal membrane. Hitherto reported transcription profiles on host-pathogen interactions are strongly biased towards in vitro studies. To detail the local in vivo genetic response to infection, we here profiled host gene expression in a recent experimental model that assures high spatial and temporal control of uropathogenic Escherichia coli (UPEC) infection within the kidney of a live rat.ResultsTranscriptional profiling of tissue biopsies from UPEC-infected kidney tissue revealed 59 differentially expressed genes 8 h post-infection. Their relevance for the infection process was supported by a Gene Ontology (GO) analysis. Early differential expression at 3 h and 5 h post-infection was of low statistical significance, which correlated to the low degree of infection. Comparative transcriptomics analysis of the 8 h data set and online available studies of early local infection and inflammation defined a core of 80 genes constituting a "General tissue response to early local bacterial infections". Among these, 25% were annotated as interferon-γ (IFN-γ) regulated. Subsequent experimental analyses confirmed a systemic increase of IFN-γ in rats with an ongoing local kidney infection, correlating to splenic, rather than renal Ifng induction and suggested this inter-organ communication to be mediated by interleukin (IL)-23. The use of comparative transcriptomics allowed expansion of the statistical data handling, whereby relevant data could also be extracted from the 5 h data set. Out of the 31 differentially expressed core genes, some represented specific 5 h responses, illustrating the value of comparative transcriptomics when studying the dynamic nature of gene regulation in response to infections.ConclusionOur hypothesis-free approach identified components of infection-associated multi-cellular tissue responses and demonstrated how a comparative analysis allows retrieval of relevant information from lower-quality data sets. The data further define marked representation of IFN-γ responsive genes and a prompt inter-organ communication as a hallmark of an early local tissue response to infection.

Project description:Although microRNAs (miRNAs) are implicated in osteosarcoma biology and chemoresponse, miRNA prognostic models are still needed, particularly because prognosis is imperfectly correlated with chemoresponse. Formalin-fixed, paraffin-embedded tissue is a necessary resource for biomarker studies in this malignancy with limited frozen tissue availability.We performed miRNA and mRNA microarray formalin-fixed, paraffin-embedded assays in 65 osteosarcoma biopsy and 26 paired post-chemotherapy resection specimens and used the only publicly available miRNA dataset, generated independently by another group, to externally validate our strongest findings (n = 29). We used supervised principal components analysis and logistic regression for survival and chemoresponse, and miRNA activity and target gene set analysis to study miRNA regulatory activity.Several miRNA-based models with as few as five miRNAs were prognostic independently of pathologically assessed chemoresponse (median recurrence-free survival: 59 months versus not-yet-reached; adjusted hazards ratio = 2.90; P = 0.036). The independent dataset supported the reproducibility of recurrence and survival findings. The prognostic value of the profile was independent of confounding by known prognostic variables, including chemoresponse, tumor location and metastasis at diagnosis. Model performance improved when chemoresponse was added as a covariate (median recurrence-free survival: 59 months versus not-yet-reached; hazard ratio = 3.91; P = 0.002). Most prognostic miRNAs were located at 14q32 - a locus already linked to osteosarcoma - and their gene targets display deregulation patterns associated with outcome. We also identified miRNA profiles predictive of chemoresponse (75% to 80% accuracy), which did not overlap with prognostic profiles.Formalin-fixed, paraffin-embedded tissue-derived miRNA patterns are a powerful prognostic tool for risk-stratified osteosarcoma management strategies. Combined miRNA and mRNA analysis supports a possible role of the 14q32 locus in osteosarcoma progression and outcome. Our study creates a paradigm for formalin-fixed, paraffin-embedded-based miRNA biomarker studies in cancer.

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Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing.

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Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing.

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