P. aeruginosa rhamnolipids stabilize human rhinovirus 14 virions

Abstract

Many mammalian viruses encounter bacteria and bacterial molecules over the course of infection. Previous work has shown that the microbial ecology of the gut plays an integral role in poliovirus and coxsackievirus infection, where bacterial glycans can facilitate virus-receptor interactions, enhance viral replication, and stabilize viral particles. However, how airway bacteria alter respiratory viral infection is less understood. Therefore, we investigated whether a panel of airway bacteria affect rhinovirus stability. We found that Pseudomonas aeruginosa, an opportunistic airway pathogen, protects human rhinovirus 14 from acid or heat inactivation. Further investigation revealed that P. aeruginosa rhamnolipids, glycolipids with surfactant properties, are necessary and sufficient for stabilization of rhinovirus virions. Taken together, this work demonstrates that specific molecules produced by an opportunistic airway pathogen can influence a respiratory virus.

Figure 1.. P. aeruginosa stabilizes HRV14.

A/B/C) HRV14 (10⁵ PFU) was incubated for one hour at a pH of 6.8 (A) or 5.8 (B) or for two hours at 33°C or 49°C (C) in the presence or absence of a panel of airway bacteria (10⁶-10⁸ CFU). Samples were centrifuged and PFU were quantified from the supernatant by plaque assay. D) HRV14 was incubated in the presence or absence of P. aeruginosa PAO1, PA14, or FRD1 at 33°C or 49°C for two hours prior to plaque assay. n=6, 3 biological replicates with 2 technical replicates. **, p<0.01, ***, p<0.001, ****, p<0.0001 (A-D Kruskal-Wallis, Dunnett’s post hoc test, B insert unpaired t test).

Figure 2.. HRV14 does not have enhanced binding to P. aeruginosa.

A/B) ³⁵S-radiolabeled HRV14 (~4,000 CPM/10⁶ PFU) was incubated in the presence or absence of streptavidin beads (2.8 µm) or 10⁶-10⁸ CFU bacteria in media at a pH of 6.8 (A) or 5.8 (B) at 33°C for one hour. Samples were centrifuged and washed to remove unbound virus. Bound virus was quantified via scintillation counting and normalized to input. n=3. A) ns, p>0.05 (Kruskal-Wallis, Dunnett’s post hoc test). B) *, p<0.05, **, p<0.01 (one-way ANOVA, Dunnett’s post hoc test).

Figure 3.. Heat-killed P. aeruginosa stabilizes HRV14.

A/B) HRV14 (10⁵ PFU) was incubated in the presence or absence of 10⁸ CFU live or heat-killed (HK) P. aeruginosa PAO1 at a pH of 5.8 or 6.8 at 33°C for one hour (A) or 33°C or 49°C for two hours (B) prior to plaque assay. C/D) HRV14 was incubated in the presence or absence of 1 mg/ml LPS from E. coli or P. aeruginosa at a pH of 5.8 or 6.8 for one hour (C) or 33°C or 49°C for two hours (D) prior to plaque assay. n=6, 3 biological replicates with 2 technical replicates. *, p<0.05, ****, p<0.0001 (A/C/D, Kruskal-Wallis, Dunnett’s post hoc test, B, one way ANOVA, Dunnett’s post hoc test).

Figure 4.. Insertion mutation of rhamnolipid synthesis genes ablates HRV14 stabilization.

A) P. aeruginosa rhamnolipid synthesis pathway. B/C) HRV14 (10⁵ PFU) was incubated in the presence or absence of 10⁸ CFU PAO1, rhlA, rhlB, or rhlC transposon insertion mutants at a pH of 5.8 or 6.8 at 33°C for one hour (B) or 33°C or 49°C for two hours (C) prior to plaque assay. n=6–8, 3–4 biological replicates with 2 technical replicates. *, p<0.05, ** p<0.01, ****, p<0.0001 (Kruskal-Wallis, Dunnett’s post hoc test).

Figure 5.. Rhamnolipids stabilize HRV14.

A/B) HRV14 (10⁵ PFU) was incubated in the presence or absence of various concentrations of rhamnolipids at a pH of 5.8 or 6.8 at 33°C for one hour (A) or 33°C or 49°C for two hours (B) prior to plaque assay. n=6, 3 biological replicates with 2 technical replicates. *, p<0.05, ** p<0.01, ***, p<0.001 ****, p<0.0001(Kruskal-Wallis, Dunnett’s post hoc test).

Figure 6.. Rhamnolipids enhance HRV14 thermostability.

HRV14 thermostability profile using a cell-free Particle Stability Thermal Release assay (PaSTRy). HRV14 (10⁵ PFU) was added to SYBR green II with or without LPS or rhamnolipids. Samples were heated from 25°C to 95°C on a 1% stepwise gradient with fluorescence monitoring. n=6, 3 biological replicates with 2 technical replicates. **, p<0.01, ****, p<0.0001 (one-way ANOVA, Dunnett’s post hoc test).