Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

Abstract

Secondary infections with Streptococcus pneumoniae cause severe pneumonia and enhance lethality during influenza epidemics and pandemics. Structural and functional similarities with viral neuraminidase (NA) suggest that the highly prevalent pneumococcal NAs, NanA and NanB, might contribute to this lethal synergism by supporting viral replication and that dual acting NA inhibitors (NAIs) will disrupt it. To verify this hypothesis, NanA and NanB were expressed in E. coli. After confirming their activity in enzyme assays, in vitro models with influenza virus A/Jena/8178/09 (Jena/8178) and the recombinant NanA or NanB (rNanA and rNanB) were established in A549 and MDCK cells to mimic the role of these pneumococcal NAs during co-infection. Studies on the influence of both NAs on viral receptor expression, spread, and yield revealed a distinct effect of NanA and NanB on viral replication in these in vitro models. Both enzymes were able to support Jena/8178 replication at certain concentrations. This synergism was disrupted by the NAIs oseltamivir, DANA, katsumadain A, and artocarpin exerting an inhibitory effect on viral NA and NanA. Interestingly, katsumadain A and artocarpin inhibited rNanA and rNanB similarly. Zanamivir did not show activity. These results demonstrate a key role of pneumococcal NAs in the lethal synergism with influenza viruses and reveal opportunities for its effective disruption.

Keywords: antibacterial; antiviral; co-pathogenesis; enzyme inhibition; microbial communication; pandemic influenza; pneumococci; secondary pneumococcal pneumonia.

FIGURE 1

Spread of A(H1N1)pdm09 strain A/Jena/8178/09 (Jena/8178) in the absence and the presence of different dilutions of recombinant NanA and NanB. The effect of NanA (A) and NanB (B) on virus spread in A549 cells was analyzed by immunocytochemical staining of viral nucleoprotein (shown in red) 48 h after infection with Jena/8178 at MOI of 0.1 TCID₅₀/cell.

FIGURE 2

Influence of recombinant NanA and NanB on expression of SA on the surface of A549 cells. Cells were treated with different neuraminidase dilutions for 48 h. The lectins MAA (A) and SNA (B) were used to detect SAα2-3Gal and SAα2-6Gal, respectively, by immunocytochemical staining. Control (Co) was stained without using lectins.

FIGURE 3

Inhibition of replication of A(H1N1)pdm09 strain A/Jena/8178/09 (Jena/8178) by neuraminidase inhibitors (NAIs). A549 cells infected with Jena/8178 (MOI of 0.1 TCID₅₀/cells) were treated with oseltamivir, zanamivir, DANA, artocarpin, and katsumadain A in absence of pneumococcal NAs (A), presence of rNanA (B) or presence of rNanB (C). Virus-infected cells were detected by immunocytochemical staining of viral nucleoprotein (shown in red) 48 h after infection to visualize the inhibitor effect on virus spread. To analyze the effect of NAIs on virus yield (D), virus titers in pfu/mL were determined with plaque assay 48 h after infection. Virus control titer was set to 100% and inhibition of the control titer by NAIs in % was calculated. Experiments were performed at least three times, and one representative assay is exemplarily shown. Significant values were calculated with non-parametric Wilcoxon–Mann–Whitney test (^∗p < 0.5, ^∗∗p < 0.1).

FIGURE 4

Computational models of the binding of oseltamivir (carbons blue) and zanamivir (carbons green) to A/Jena/8178/09, NanA and NanB. While both inhibitors fit well to the viral NA (A), steric clashes (marked by a red X) of zanamivir with I416 in NanA (B) and the equivalent in NanB, I326, and T539 (C) are apparent. These are likely the cause for the observed inactivity of zanamivir on both bacterial NAs.