Systematic analysis of proximal midgut- and anorectal-originating contractions in larval zebrafish using event feature detection and supervised machine learning algorithms.

BACKGROUND: Zebrafish larvae are translucent, allowing in vivo analysis of gut development and physiology, including gut motility. While recent progress has been made in measuring gut motility in larvae, challenges remain which can influence results, such as how data are interpreted, opportunities for technical user error, and inconsistencies in methods. METHODS: To overcome these challenges, we noninvasively introduced Nile Red fluorescent dye to fill the intraluminal gut space in zebrafish larvae and collected serial confocal microscopic images of gut fluorescence. We automated the detection of fluorescent-contrasted contraction events against the median-subtracted signal and compared it to manually annotated gut contraction events across anatomically defined gut regions. Supervised machine learning (multiple logistic regression) was then used to discriminate between true contraction events and noise. To demonstrate, we analyzed motility in larvae under control and reserpine-treated conditions. We also used automated event detection analysis to compare unfed and fed larvae. KEY RESULTS: Automated analysis retained event features for proximal midgut-originating retrograde and anterograde contractions and anorectal-originating retrograde contractions. While manual annotation showed reserpine disrupted gut motility, machine learning only achieved equivalent contraction discrimination in controls and failed to accurately identify contractions after reserpine due to insufficient intraluminal fluorescence. Automated analysis also showed feeding had no effect on the frequency of anorectal-originating contractions. CONCLUSIONS & INFERENCES: Automated event detection analysis rapidly and accurately annotated contraction events, including the previously neglected phenomenon of anorectal contractions. However, challenges remain to discriminate contraction events based on intraluminal fluorescence under treatment conditions that disrupt functional motility.

The E. coli transcription factor GrlA is regulated by subcellular compartmentalization and activated in response to mechanical stimuli.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that colonizes the gastrointestinal tract and has evolved intricate mechanisms to sense and respond to the host environment. Upon the sensation of chemical and physical cues specific to the host's intestinal environment, locus of enterocyte effacement (LEE)-encoded virulence genes are activated and promote intestinal colonization. The LEE transcriptional activator GrlA mediates EHEC's response to mechanical cues characteristic of the intestinal niche, including adhesive force that results from bacterial adherence to epithelial cells and fluid shear that results from intestinal motility and transit. GrlA expression and release from its inhibitor GrlR was not sufficient to induce virulence gene transcription; mechanical stimuli were required for GrlA activation. The exact mechanism of GrlA activation, however, remained unknown. We isolated GrlA mutants that activate LEE transcription, independent of applied mechanical stimuli. In nonstimulated EHEC, wild-type GrlA associates with cardiolipin membrane domains via a patch of basic C-terminal residues, and this membrane sequestration is disrupted in EHEC that expresses constitutively active GrlA mutants. GrlA transitions from an inactive, membrane-associated state and relocalizes to the cytoplasm in response to mechanical stimuli, allowing GrlA to bind and activate the LEE1 promoter. GrlA expression and its relocalization in response to mechanical stimuli are required for optimal virulence regulation and colonization of the host intestinal tract during infection. These data suggest a posttranslational regulatory mechanism of the mechanosensor GrlA, whereby virulence gene expression can be rapidly fine-tuned in response to the highly dynamic spatiotemporal mechanical profile of the gastrointestinal tract.

The zebrafish as a model for gastrointestinal tract-microbe interactions.

The zebrafish (Danio rerio) has become a widely used vertebrate model for bacterial, fungal, viral, and protozoan infections. Due to its genetic tractability, large clutch sizes, ease of manipulation, and optical transparency during early life stages, it is a particularly useful model to address questions about the cellular microbiology of host-microbe interactions. Although its use as a model for systemic infections, as well as infections localised to the hindbrain and swimbladder having been thoroughly reviewed, studies focusing on host-microbe interactions in the zebrafish gastrointestinal tract have been neglected. Here, we summarise recent findings regarding the developmental and immune biology of the gastrointestinal tract, drawing parallels to mammalian systems. We discuss the use of adult and larval zebrafish as models for gastrointestinal infections, and more generally, for studies of host-microbe interactions in the gut.