SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus

Abstract

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike-biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.

Keywords: COVID-19; MHV; S1 and S2 subunits; SARS-CoV-2; Staphylococcus aureus; Streptococcus pneumoniae; biofilm; coronavirus; nucleocapsid; spike protein.

Figure 1

Effect of initial bacterial cell density on biofilm production by S. pneumoniae 19F and S. aureus A10 strains using the crystal violet assay. Each result was derived from three independent experiments or biological replicates (with each assay performed as technical triplicates). Each small gray datapoint represents the average or mean of technical triplicates, while each blue datapoint depicts the mean of three independent experiments. Intervals represent 95% confidence interval (CI) for the mean. p-value < 0.05 was considered statistically significant by the independent samples Mann-Whitney U test.

Figure 2

Characterization of biofilm production by (a) S. pneumoniae 19F and (b) S. aureus A10 strains, when co-incubated with recombinant SARS-CoV-2 spike S1 subunit, S2 ECD, S1 + S2 subunits, and nucleocapsid protein (NP) using the crystal violet assay. Each result was derived from three independent experiments or biological replicates (with each assay performed as technical triplicates). Each small gray datapoint represents the average or mean of technical triplicates, while each blue datapoint depicts the mean of three independent experiments. Intervals represent 95% CI for the mean. p-value < 0.05 was considered statistically significant by one-way ANOVA with Dunnett’s T3 post-hoc test.

Figure 2

Characterization of biofilm production by (a) S. pneumoniae 19F and (b) S. aureus A10 strains, when co-incubated with recombinant SARS-CoV-2 spike S1 subunit, S2 ECD, S1 + S2 subunits, and nucleocapsid protein (NP) using the crystal violet assay. Each result was derived from three independent experiments or biological replicates (with each assay performed as technical triplicates). Each small gray datapoint represents the average or mean of technical triplicates, while each blue datapoint depicts the mean of three independent experiments. Intervals represent 95% CI for the mean. p-value < 0.05 was considered statistically significant by one-way ANOVA with Dunnett’s T3 post-hoc test.

Figure 3

Characterization of biofilm production by S. pneumoniae 19F and S. aureus A10 strains when co-incubated with murine hepatitis virus (MHV) or cell-free culture supernatant (control) using the crystal violet assay. Each result was derived from three independent experiments or biological replicates (with each assay performed as technical triplicates). Each small gray datapoint represents the average or mean of technical triplicates, while each blue datapoint depicts the mean of three independent experiments. Intervals represent 95% CI for the mean. p-value < 0.05 was considered statistically significant by one-way ANOVA with Dunnett’s T3 post-hoc test.

Figure 4

Particle-tracking microrheology analysis of biofilms of (a,b) S. pneumoniae and (c,d) S. aureus. (a,c) Creep compliance J versus lag-time t at 296 K corresponding to different beads were measured at randomly chosen locations in each sample. (b,d) Shown are the elastic storage modulus $G\prime$ (red) and viscous loss modulus $G″$ (blue) versus frequency $\omega$, corresponding to the highest (bottom pair) and lowest (top pair) $J\left(t\right)$, respectively.

Figure 5

Effect of co-incubating SARS-CoV-2 spike S1 and S2 antibodies with spike S1 + S2 protein on biofilm production by S. pneumoniae 19F assessed by the crystal violet assay. The dilutions of both anti-S1 and anti-S2 antibodies used are denoted as 1:10,000 and 1:1000. Spike antibodies (Ab) were co-incubated with SARS-CoV-2 recombinant spike S1 + S2 subunits (10 pmol) for 1 h. Bacteria were then added, and further co-incubated for 18 h for biofilm formation. In the control, no spike antibodies nor proteins were co-incubated with bacteria. Co-incubations of spike antibodies only with bacteria were also tested as controls. Each result was derived from three independent experiments or biological replicates (with each assay performed as technical triplicates). Each small gray datapoint represents the average or mean of technical triplicates, while each blue datapoint depicts the mean of three independent experiments. Intervals represent 95% CI for the mean. p-value < 0.05 was taken as statistically significant by one-way ANOVA with Dunnett’s T3 post-hoc test.

Figure 6

Western blot detection of recombinant SARS-CoV-2 spike S1 + S2 subunits bound to S. pneumoniae biofilm (using whole bacterial cell lysate). The blots were individually probed with spike S1 and S2 antibodies. The predicted molecular masses of recombinant spike S1, S2 ECD, and S1 + S2 proteins are 76, 59, and 134 kDa, respectively. Lane 1: Control of S. pneumoniae only; lane 2: S. pneumoniae incubated with S1 + S2 subunits. The results of biological duplicate experiments are shown.