Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae

Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells – termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria haemocytes, but protocols include both centrifugation and fixation – physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that Galleria haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall β-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.</p

Impact of catalase on resistance to neutrophil killing.

<p><i>C</i>. <i>albicans</i> wild-type (<i>CAT1</i>, black, Ca2084), <i>cat1</i>Δ (red, Ca Ca2089) and <i>tetON-CAT1</i> cells (Ca2038) pre-grown with 0 or 20 with μM doxycycline (pale blue and blue, respectively) were exposed to human neutrophils for 2 h, and then fungal survival assayed. Each data point represents the mean for three replicates from one healthy donor. The data were analysed using one-way ANOVA with Tukey’s post-hoc test: *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01.</p

Manipulation of catalase levels in <i>C</i>. <i>albicans</i>.

<p>Catalase activities were measured in protein extracts from mid-exponential <i>C</i>. <i>albicans</i> cultures containing 0 or 20 μM doxycycline (- or + Dox, respectively): <i>cat1</i>Δ, Ca2089; wild-type, WT, Ca2084; red, <i>tetON-CAT1</i> isolates, Ca2040, Ca2043, Ca2046 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). Immediately before harvesting, wild-type and <i>cat1</i>Δ cultures were exposed to 0 or 5 mM H₂O₂ for one hour. Means and standard deviations from three independent replicate experiments are shown, and the data were analysed using one-way ANOVA with Tukey’s post-hoc test: *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001; ****, <i>p</i> ≤ 0.0001.</p

In the absence of stress, elevated catalase levels impose a fitness defect on <i>C</i>. <i>albicans</i> that is suppressed by iron supplementation.

<p><b>(A)</b> The growth of <i>C</i>. <i>albicans tetON-CAT1</i> isolates (1, 4, 10) was monitored (OD₆₀₀) in YPD containing 0 or 20 μM doxycycline (Ca2040, Ca2043, Ca2046; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). The impact of <i>tetON-CAT1</i> induction upon fitness was assayed by subtracting the OD after growth in the absence of doxycycline (OD_-Dox) from the OD in the presence of doxycycline (OD_+Dox). Data represent the means and standard deviations from three independent experiments. <b>(B)</b> The impact of iron on the fitness of <i>C</i>. <i>albicans tetON-CAT1</i> strain 1 (Ca2040) was measured in YPD cultures containing 0 or 20 μM doxycycline plus different concentrations of FeCl₃. Data represent the means and standard deviations from three independent experiments. <b>(C)</b> The effect of <i>tetON-CAT1</i> expression on genes involved in iron assimilation and homeostasis was assessed by qRT-PCR of specific transcripts (relative to the <i>ACT1</i> mRNA internal control) during growth of <i>C</i>. <i>albicans tetON-CAT1</i> isolate 1 (Ca2040) in YPD containing 0 or 20 μM doxycycline. The data, which represent the means and standard deviations from three replicate measurements, are normalised relative to the corresponding transcript level in wild type cells grown with doxycycline.</p

Stochastic differences in catalase expression within a population of <i>C</i>. <i>albicans</i> cells affect resistance to peroxide stress.

<p><b>(A)</b> Western blot of GFP in <i>C</i>. <i>albicans</i> cells grown in YPD at 30°C: GFP, cells expressing GFP from pACT1-GFP (Ca230); <i>CAT1</i>, control cells with no GFP (Ca674); <i>CAT1-GFP</i>, cells expressing Cat1-GFP (Ca2213) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). <b>(B)</b> DIC and fluorescence microscopy of log-phase <i>CAT1-GFP</i> cells (Ca2213) exposed to 0 or 5 mM H₂O₂ for 1 h: scale bar = 5 μm. <b>(C)</b> The resistance of <i>C</i>. <i>albicans</i> strains to peroxide was assessed by plating onto YPD plates containing 0, 5 or 10 mM H₂O₂: <i>CAT1/CAT1</i>, Ca674; <i>CAT1/cat1</i>Δ, Ca1862; <i>cat1</i>Δ/<i>cat1</i>Δ, Ca1864; <i>CAT1-GFP</i>, Ca2213. For each stress condition, the images for all strains were taken from the same plate. <b>(D)</b> Sorting of <i>C</i>. <i>albicans</i> cells of similar size expressing relatively low or high levels of Cat1-GFP within the same population growing on YPD (no stress) by FACS: Cat1-GFP cells, Ca2213; No GFP control, Ca674. <b>(E)</b> The survival of FACS sorted cells with low or high Cat1-GFP levels on YPD plates containing different concentrations of H₂O₂ (CFU) expressed as a percentage of the survival for the no stress control. Means and standard deviations from three replicates are presented: *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001; ****, <i>p</i> ≤ 0.0001.</p

Impact of catalase levels on host colonisation during systemic infection.

<p>The nine <i>C</i>. <i>albicans</i> strains were grown separately in medium containing 0 or 20 μM doxycycline, mixed in approximately equal proportions, and then this pool of nine barcoded strains used to initiate systemic infection in mice via the tail vein (n = 6 mice per group): wild-type (<i>CAT1</i>) strains 23, 21, 26 (Ca2084, Ca2085, Ca2087); <i>cat1</i>Δ strains 28, 38, 54 (Ca2089, Ca2092, Ca2030); <i>tetON-CAT1</i> strains 1, 4, 10, (Ca2038, Ca2041, Ca2044; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). Mice that received the pool of nine <i>C</i>. <i>albicans</i> strains pre-grown with doxycycline were treated with doxycycline, whereas mice that were infected with the pool of <i>C</i>. <i>albicans</i> strains pre-grown without doxycycline did not (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#sec011" target="_blank">Materials and Methods</a>). After four days, mice were killed by cervical dislocation and the kidneys, brains, livers and spleens harvested. The fungal cells infecting these organs were grown on YPD plates, genomic DNA isolated from each fungal population, and the abundance of each barcode in each fungal population was quantified by barseq. The Relative Abundance of each barcode in each tissue from each mouse was calculated relative to the total number of barcode reads for that specific sample, and then normalised against the proportion for that barcode in the pool of nine <i>C</i>. <i>albicans</i> strains used to infect that mouse. Each symbol represents data for a single mouse. The pairwise comparisons indicated were analysed using the Students t-test: *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01. Data are presented for three of the nine barcoded <i>C</i>. <i>albicans</i> strains: <i>CAT1</i> strain 23, grey and black; <i>cat1</i>Δ strain 28, pink and red; <i>tetON-CAT1</i> strain 1, pale blue and blue; no doxycycline, grey, pink, pale blue; 20 μM doxycycline, black, red, blue. These wild type and <i>cat1</i>Δ strains were representative of the other isolates. However, <i>tetON-CAT1-</i>1 behaved differently from the other <i>tetON-CAT1</i> isolates, which were shown subsequently to have lost their phenotype (below).</p

Inactivation of catalase does not attenuate <i>C</i>. <i>albicans</i> virulence.

<p><b>(A)</b> The virulence of <i>C</i>. <i>albicans</i> wild-type (<i>CAT1</i>, Ca2084) and <i>cat1</i>Δ (Ca2089) strains was first tested in a short term model of systemic infection. Mice were infected with similar doses of either <i>CAT1</i> or <i>cat1</i>Δ cells via their tail vein, and their weights monitored over time. Animals were sacrificed after four days, their weight change determined and their kidney fungal burdens assayed. <b>(B)</b> The same strains were compared in a longer term mouse model of systemic infection. Mouse survival data are presented in a Kaplan-Meier plot: the differences are not statistically significant (<i>p</i> = 0.074). <b>(C)</b> <i>C</i>. <i>albicans</i> wild-type (<i>CAT1</i>, Ca2084), <i>cat1</i>Δ (Ca2089) and <i>tetON-CAT1</i> strains (Ca2038) were compared in the <i>Galleria mellonella</i> model of systemic infection. As a negative control, larvae were injected with phosphate buffered saline. Survival data are presented in a Kaplan-Meier plot. The differences between the <i>C</i>. <i>albicans</i> strains are not statistically significant (<i>p</i> = 0.68).</p

Impact of catalase levels upon <i>C</i>. <i>albicans</i> fitness in the presence or absence of peroxide stress.

<p>The nine barcoded <i>C</i>. <i>albicans</i> wild-type, <i>cat1</i>Δ and <i>tetON-CAT1</i> strains (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>) were pre-grown separately in YPD containing 0 or 20 μM doxycycline. The cells grown in the presence of doxycycline were then mixed in roughly equal proportions and used to inoculate fresh YPD cultures containing doxycycline (plus Dox). In parallel, the cells grown in the absence of doxycycline were mixed and used to inoculate fresh YPD cultures lacking doxycycline (no Dox). Parallel cultures contained 0 or 5 mM H₂O₂ (no stress and plus stress, respectively). Cells were harvested at various time points over a 12 h period, and barseq was performed on genomic DNA extracted from each sample. The relative abundance of each barcode in each sample was then calculated relative to its starting abundance. Data are shown for one barcoded strain of each type (means and standard deviation from three replicate measurements): wild-type (<i>CAT1</i>), black, Ca2084; <i>cat1</i>Δ, red, Ca2089; <i>tetON-CAT1</i>, blue, Ca2038. <b>(A)</b> Competition between wild-type, <i>cat1</i>Δ and <i>tetON-CAT1</i> strains in the absence of doxycycline and the absence of peroxide stress. <b>(B)</b> Competition in the absence of doxycycline and the presence of stress. <b>(C)</b> Competition in the presence of doxycycline and the absence of stress. <b>(D)</b> Competition in the presence of both doxycycline and stress.</p

Elevated basal levels of catalase protect against oxidative and combinatorial stresses.

<p><b>(A)</b> Dilutions of mid-exponential <i>C</i>. <i>albicans</i> cultures were spotted onto YPD plates containing different stresses and photographed after 24 h growth at 30°C: control; 1 M NaCl; 7.5 mM H₂O₂; or 5 mM H₂O₂ plus 1 M NaCl. Strains were either grown in the absence of doxycycline (left panels; see cartoons above the panels), pre-grown without doxycycline and then plated onto media containing 20 μM doxycycline (central panels), or pre-grown with 20 μM doxycycline and then plated onto media lacking doxycycline (right panels): wild-type <i>CAT1</i> isolates 23, 21, 26 (Ca2084, Ca2085, Ca2087); <i>cat1</i>Δ isolates 28, 38, 54 (Ca2089, Ca2092, Ca2030); <i>tetON-CAT1</i> isolates 1, 4, 10 (Ca2038, Ca2041, Ca2044; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). These data reflect the outputs of three independent experiments. <b>(B)</b> Mid-exponential wild type (<i>CAT1</i>, <i>Ca</i>2084), <i>cat1</i>Δ (Ca2089) and <i>tetON-CAT1</i> cells (Ca2038) grown in YPD containing 20 μM doxycycline were treated with 5 mM H₂O₂ and/or 1 mM NaCl for 1 h. Cells were stained with propidium iodide (PI) and the percentage of PI positive (dead) cells quantified by flow cytometry. Means and standard deviations from three replicates are presented: *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001. <b>(C)</b> Wild type (<i>CAT1</i>, Ca2084), <i>cat1</i>Δ (Ca2089) and <i>tetON-CAT1</i> cells (Ca2038) grown in YPD plus 20 μM doxycycline were treated with 5 mM H₂O₂ and/or 1 mM NaCl for 1 h, and their accumulation of intracellular ROS quantified by flow cytometry. Means and standard deviations from three replicates are presented: *, <i>p</i> ≤ 0.05.</p

Impact of catalase on resistance to neutrophil killing.

<p><i>C</i>. <i>albicans</i> wild-type (<i>CAT1</i>, black, Ca2084), <i>cat1</i>Δ (red, Ca Ca2089) and <i>tetON-CAT1</i> cells (Ca2038) pre-grown with 0 or 20 with μM doxycycline (pale blue and blue, respectively) were exposed to human neutrophils for 2 h, and then fungal survival assayed. Each data point represents the mean for three replicates from one healthy donor. The data were analysed using one-way ANOVA with Tukey’s post-hoc test: *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01.</p