The in vitro and in vivo effects of constitutive light expression on the mouse enteropathogen Citrobacter rodentium
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Abstract
Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we investigated whether constitutive light expression makes the bioluminescent C. rodentium strain ICC180 less competitive when competed against its non-bioluminescent parent (strain ICC169). To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggests that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.
Cite this as
2016. The in vitro and in vivo effects of constitutive light expression on the mouse enteropathogen Citrobacter rodentium. PeerJ Preprints 4:e1993v1 https://doi.org/10.7287/peerj.preprints.1993v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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This is a submission to PeerJ for review.
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Supplemental Information
Raw data of bacterial counts, bioluminescence and growth in vitro and in vivo
Elbow tests of phenotypic microarray array data to determine the number of clusters appropriate for k-means clustering
Data was analysed using the DuctApe software suite.
Supplementary Fig. 2. The growth of C. rodentium ICC180 compared to its non-bioluminescent parent strain ICC169 as assessed by phenotypic microarray (PM)
Wildtype C. rodentium ICC169 (shown as purple lines) and its bioluminescent derivative ICC180 (shown as blue lines) were grown on two separate occasions using PM plates 1-20 (categorised by colour [see Key]). Differences between the growth of ICC169 and ICC180 in each individual well were analysed using the moderated t-test provided by limma . Wells in which the differences had an adjusted p-value of less than 0.5 (stringent cut-off) are shown.
Supplementary Fig. 3. Infection of larvae of the Greater Wax Moth Galleria mellonella with bioluminescent C. rodentium ICC180 can be visualised by luminometry
Groups of larvae (n = 10) of the Greater Wax Moth Galleria mellonella were infected with ~108 CFU of C. rodentium ICC169 or ICC180 and monitored for bioluminescence using a plate luminometer. Data (medians with ranges) is presented from experiments performed on 3 separate occasions and is given as relative light units [RLU] waxworm-1.
Additional Information
Competing Interests
Siouxsie Wiles is an Academic Editor for PeerJ.
Author Contributions
Hannah M Read conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Grant Mills performed the experiments, reviewed drafts of the paper.
Sarah Johnson performed the experiments, reviewed drafts of the paper.
Peter Tsai analyzed the data, prepared figures and/or tables, reviewed drafts of the paper.
James Dalton performed the experiments, analyzed the data, reviewed drafts of the paper.
Lars Barquist analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Cristin G Print contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Wayne M Patrick analyzed the data, reviewed drafts of the paper.
Siouxsie Wiles conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
Experiments were performed in accordance with the New Zealand Animal Welfare Act (1999) and institutional guidelines provided by the University of Auckland Animal Ethics Committee, which reviewed and approved these experiments under applications R1003 and R1496.
Funding
This work was supported by seed funding from the Maurice Wilkins Centre for Molecular Biodiscovery, and by a Sir Charles Hercus Fellowship to SW (09/099) from the Health Research Council of New Zealand. LB is supported by a Research Fellowship from the Alexander von Humboldt Stiftung/Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
