Microbiology and Infectious Disease

Bacteria Are a Major Determinant of Orsay Virus Transmission and Infection in Caenorhabditis elegans

Brian G. Vassallo
Noémie Scheidel
Sylvia E. J. Fischer
Dennis H. Kim author has email address

Division of Infectious Diseases, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School; Boston, 02115, USA
Department of Biology, Massachusetts Institute of Technology; Cambridge, 02139, USA

https://doi.org/10.7554/eLife.92534.1

Open access
Copyright information

Figures and data

Ochrobactrum species and P. lurida MYb11 divergently modulate Orsay virus transmission and infection rates.
(A) Schematic representation of the transmission and susceptibility assays: Transmission can be assessed by combining infected spreader individuals (green nematode), uninfected reporter individuals (white nematodes), and various bacteria. Susceptibility can be assessed by combining uninfected reporter individuals with exogenous Orsay virus, and various bacteria. 24 h later, infection is assessed. This schematic was made using Biorender. (B) Representative images of GFP expression among individuals exposed to spreaders or no spreaders in the presence of O. vermis MYb71, E. coli OP50, or P. lurida MYb11. (C) Incidence proportion, calculated as indicated, of Orsay virus transmission quantified on different bacteria from the environment of C. elegans. Data shown are from three experiments combined, each dot represents the incidence proportion from a single plate. (D) Dose response curves of C. elegans to exogenous Orsay virus. The dashed line indicates the calculated dose at which 50% of the population was infected, the ID₅₀, the exact value which is given above the x-axis for each bacterium. The solid curves represent the 95% confidence interval of the modeled log-logistic function while in the presence of each bacterium. Data are from a single representative experiment, Dots represent individual plates. ((a.u.), arbitrary units). (E) Table shows the mean +/- the standard deviation of the ID₅₀ ratio of the indicated bacteria measured in three experiments as in (D). (F) The fraction of individuals with staining as assessed by Fluorescence in situ hybridization targeting the RNA1 segment of Orsay virus. Data shown are from three experiments combined, each dot represents three pooled technical replicates from a susceptibility assay as in (D). For all plots the black bar is the mean and error bars are the 95% confidence interval (C.I.). p-values determined using one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) test (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

P. aeruginosa attenuates Orsay virus transmission and infection rates.
(A) Incidence proportion of Orsay virus transmission quantified on different bacteria from C. elegans’ environment and P. aeruginosa PA01 and P. aeruginosa PA14. Data shown are from three experiments combined, each dot represents the incidence proportion from a single plate. (B) The fraction of individuals GFP positive following exposure to two doses of exogenous Orsay virus. Data are from a single representative experiment and dots represent individual plates. (C) The fraction of individuals with staining following exposure to 100a.u. Orsay Virus as assessed by fluorescence in situ hybridization targeting the RNA1 segment of the Orsay Virus genome. Data shown are from three experiments combined, each dot represents three pooled technical replicates from an experiment. For all plots the black bar is the mean and error bars are the 95% confidence interval (C.I.). p-values determined using one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) test (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).

P. aeruginosa and P. lurida MYb11 do not eliminate Orsay virus replication.
(A) Animals carrying either a wild-type (RNA1(WT)) or replication defective (RNA1(D601A)) transgenic viral RNA replicon system were used to assess Orsay virus replication efficiency independently of virus entry. P-values determined using one-way ANOVA followed by Dunnett’s test (B) Orsay virus RNA1 levels of N2 and rde-1(ne219) animals exposed to P. aeruginosa PA14 and exogenous Orsay virus 2- and 24-h post infection (hpi). P-values were determined using Welch’s t-Test For each plot, each dot represents five pooled technical replicates, the black bar is the mean, error bars are the standard deviation, RNA1 levels were quantified by qPCR, and the data shown are for three independent experiments. (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).

Mutation of P. aeruginosa quorum sensing regulators and the two-component response regulator gacA suppresses the attenuation Orsay virus infection.
(A) Diagram demonstrating the three quorum sensing systems in P. aeruginosa. Each system encodes the enzyme(s) (LasI, RhlI, PqsA(BCDH) to produce an autoinducer (3OC12HSL – 3-oxo-C12-homoserine lactone, C4HSL –butanoyl homoserine lactone, PQS – Pseudomonas quinolone signal) that is recognized by its cognate receptor (LasR, RhlR, PqsR) that influences gene transcription and the activity of the other quorum sensing systems. An additional level of regulation stems from the two-component gacS/gacA system. External signals are recognized by the histidine kinase GacS, which phosphorylates the response regulator GacA that indirectly influences quorum sensing processes. IM is inner membrane, OM is outer membrane. Arrows represent crosstalk between the various system. Adapted from Rutherford and Bassler 2012 and Song, Li, and Wang 2023. (B-C) The fraction of individuals GFP positive following exposure to exogenous Orsay virus. Data are from a single representative experiment, bars represent mean, dots represent individual plates. (B) Wild-type P. aeruginosa PA01 compared to mutant P. aeruginosa PA01 strains using 10a.u. of Orsay virus. (C) Wild-type P. aeruginosa PA14 compared to mutant P. aeruginosa PA14 strains using 100a.u. of Orsay virus. (D) Incidence proportion of Orsay virus transmission in the presence of P. aeruginosa wild-type versus select P. aeruginosa mutants. Data are from three experiments, bars represent mean, dots represent individual plates. For all plots error bars represent 95% C.I. For B-E, p-values were determined using one-way ANOVA followed by Dunnett’s test. (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).

Mutation of P. lurida MYb11 gacA suppresses the attenuation of Orsay virus infection.
(A) Diagram depicting P. aeruginosa PA01, P. aeruginosa PA14, and P. lurida MYb11 gacA as well as the P. lurida MYb11 gacA deletion mutant. Percent identity of the encoded protein was assessed using Clustal Omega. The gacA deletion removed 194 amino acids from the protein leaving 10 amino acids from both the N and C termini. Schematics made using Biorender.(B) The fraction of individuals GFP positive following exposure to 10a.u. exogenous Orsay virus in the presence of wild-type P. lurida MYb11 compared to a gacA mutant. Data are from a single representative experiment, bars represent mean, dots represent individual plates. (C) Incidence proportion of Orsay virus transmission in the presence of wild-type P. lurida MYb11 compared to a gacA mutant. Data are from three experiments, bars represent mean, dots represent individual plates. For all plots error bars represent 95% confidence interval (C.I.). p-values were determined using Student’s t-Test (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Mutation of P. aeruginosa PA14 genes related to virulence suppresses the attenuation of Orsay virus infection.
(A) Diagram describing the design and results of the screen to identify suppressors of P. aeruginosa PA14 Orsay virus attenuation. (B) The fraction of individuals GFP positive following exposure to 100 a.u. of exogenous Orsay virus. Data are from a single representative experiment. (C) Incidence proportion of Orsay virus transmission while individuals are present on P. aeruginosa PA14 wild-type compared to P. aeruginosa PA14 mutants. Data are from three experiments and dots represent individual plates. For all plots the black bar is the mean and error bars are the 95% confidence interval (C.I.). p-values determined using one-way ANOVA followed by Dunnett’s test (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).

Transmission does not occur from individuals infected during the course of a transmission or susceptibility assay.
(A-B) Individuals were exposed to 0 a.u. or 5 a.u. exogenous Orsay virus in the presence of O. vermis MYb71. 8 h post infection five infected individuals from the 5 a.u. plate were transferred to the 0 a.u. plate to assess whether transmission from these individuals would occur. Both plates were scored 16 h after the transfer. Data are from a single representative experiment, bars represent mean, dots represent individual plates. Error bars are the 95% confidence interval (C.I.). p-value determined by Student’s t-test (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).

Knockout of the P. lurida MYb11 orthologs of ptsP, prpC, or kinB does not suppress Orsay virus attenuation in susceptibility or transmission assays.
(A) The fraction of individuals GFP positive following exposure to 10 a.u. of exogenous Orsay virus in the presence of the indicated bacteria. Data are from a single representative experiment. (C) Incidence proportion of Orsay virus transmission in the presence of the indicated bacteria. The E. coli OP50 reference is shared with Figure 5C. Data are from three experiments and dots represent individual plates. For all plots the black bar is the mean and error bars are the 95% confidence interval (C.I.). p-values determined using one-way ANOVA followed by Dunnett’s test (NS non-significant, *p<0.05, **p<0.01, ***p<0.001).

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