Visualization of coral host--pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain’s virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen’s interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.Human Frontier Science Program (Strasbourg, France) (No. RGY0089RS

Vibrio coralliilyticus search patterns across an oxygen gradient

The coral pathogen, Vibrio coralliilyticus shows specific chemotactic search pattern preference for oxic and anoxic conditions, with the newly identified 3-step flick search pattern dominating the patterns used in oxic conditions. We analyzed motile V. coralliilyticus cells for behavioral changes with varying oxygen concentrations to mimic the natural coral environment exhibited during light and dark conditions. Results showed that 3-step flicks were 1.4× (P = 0.006) more likely to occur in oxic conditions than anoxic conditions with mean values of 18 flicks (95% CI = 0.4, n = 53) identified in oxic regions compared to 13 (95% CI = 0.5, n = 38) at anoxic areas. In contrast, run and reverse search patterns were more frequent in anoxic regions with a mean value of 15 (95% CI = 0.7, n = 46), compared to a mean value of 10 (95% CI = 0.8, n = 29) at oxic regions. Straight swimming search patterns remained similar across oxic and anoxic regions with a mean value of 13 (95% CI = 0.7, n = oxic: 13, anoxic: 14). V. coralliilyticus remained motile in oxic and anoxic conditions, however, the 3-step flick search pattern occurred in oxic conditions. This result provides an approach to further investigate the 3-step flick.This study was funded by an ARC Discovery Grant to JG Mitchell. The associated grant number is DP1096658 with URL www.arc.gov.au. Work at the Cape Ferguson laboratories was supported by the Australian Institute of Marine Science

The measurement locations within the observation chamber.

<p>(a) Microscope slide, (b) cover slips, (c) center recordings, (d) edge recordings. A 3D numerical model depicts how oxygen diffuses from the top and the bottom of the coverslip labelled (e). Red depicts high oxygen concentrations with a vertical length of 5250 µm, whilst the blue depicts low oxygen concentrations. The chamber has a vertical length of 26,250 µm. The red dashed line illustrates the chamber cover, which sits on top of two cover slips.</p

<i>Vibrio coralliilyticus</i> search patterns along an oxygen gradient.

<p>(a) The mean total number of search patterns seen along the transect line consisting of 40 quadrat boxes (error bars = 95% CI); (b) Mean velocity (µm s⁻¹) of search patterns across the transect line (error bars = 95% CI). The location of the grey gradient lines represents the location where the oxygen transition occurs in Fig. 1 (orange and green area).</p

<i>Vibrio coralliilyticus</i> search patterns over a 25 minute time series.

<p>(a) The mean total number of search patterns seen at the edge of the cover slip and center of the cover slip over a 25 minute time series. The solid squares indicate the number of non-flick search patterns at an anoxic area of the chamber, open squares indicate the number of non-flick search patterns at an oxic area of the chamber, closed circles indicate the number of flicks search patterns at an anoxic area of the chamber, open circles indicate the number of flick search patterns at an oxic area of the chamber; (b) The mean search pattern velocities seen at oxic and anoxic regions of the cover slip chamber over a 25 minute time experiment. * = only 1 search pattern observed so no 95% CI value could be calculated; ? = missing data as no search patterns were identified.</p

Maximum, and mean search pattern speeds (µm s⁻¹) and total search pattern numbers (n) identified in oxic and anoxic regions of the observation viewing chamber.

*<p> = maximum search pattern speed.</p><p>n = number of recordings.</p

Schematic and observed chemotactic search patterns of <i>V. coralliilyticus</i>.

<p>The search pattern path starts at X; (a) cyclic 3-step flick search pattern, shown as a forward run, a reverse; seen as a 180° reorientation, and a 90° flick of the flagellum and repeat; (b) run and reverse and swimming search pattern. Run and reverse search patterns are characteristic of a 180° reorientation and reversal. Straight swimming search patterns shows no reversals; (c) 3-step flick search pattern trajectories (closed squares) collected from <i>V. coralliilyticus</i> overnight cultures collected from the oxic region of the observation chamber. For clarity, the open circles mark the 90° flicking events and the red-dashed lines indicate the 180° reversals; (d) search pattern trajectories collected from <i>V. coralliilyticus</i> overnight cultures collected from the anoxic region of the observation chamber, the search patterns exhibited here are run and reverses (open circles) and straight swimming (closed squares).</p

Three-dimensional numerical models of oxygen diffusion microscope slide chamber where oxygen diffuses from the top and the bottom.

<p>Blue dots represent bacteria that absorb oxygen as it diffuses past. The columns are 100 µm high (a and b). The relative concentration difference between blue and red is 30 mM. Orthogonalized spacings represent average cell distributions seen in the experiments with (a) for low cell density regions and (b) for cell cluster regions. Real distributions will be heterogenously rather than uniformly distributed. The diagonal in (c) is the rotated volume showing the z axis and the position of the bacteria in that direction.</p