Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases are structurally related to viral neuraminidases and susceptible to oseltamivir, an inhibitor designed to target viral neuraminidases. This prompted us to evaluate the antipneumococcal potential of two neuraminidase inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC 50 value) of the tested compounds towards pneumococcal neuraminidase.The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA neuraminidase. Unlike oseltamivir, which competes with the natural substrate of neuraminidase, artocarpin exhibits a mixed-type inhibition with a K i value of 9.70 μM. Remarkably, artocarpin was the only neuraminidase inhibitor for which an inhibitory effect in pneumococcal growth (MIC: 0.99-5.75 μM) and biofilm formation (MBIC: 1.15-2.97 μM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by * Corresponding author: Schmidtke, Michaela:

Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4′-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM, and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems.

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.

Aims: In this study, we analyze the challenges involved in detecting novel neuraminidase inhibitors (NAIs) and offer strategies to overcome them with complementary bioassays. Materials & Methods: We investigated the inhibitory activities of NAIs (oseltamivir, zanamivir, DANA, katsumadain A and remazol) as well as non-NAIs (amantadine, nucleozin and rifampicin) on influenzaviral and bacterial (Streptococcus pneumoniae, Clostridium perfringens and Vibrio cholerae) neuraminidases (NAs) with chemiluminescence (CL)- and fluorescence (FL)-based assays. Furthermore, hemagglutination-based NA inhibition assays were established. Results: Our study shows three types of signal interference affecting the readout of biochemical assays: self-FL (katsumadain A and remazol), FL quenching (rifampicin) and CL quenching (rifampicin, remazol, nucleozin and katsumadain A). These challenges were overcome by hemagglutination-based assays. Conclusion: The latter allow a robust performance in discriminating NAIs and non-NAIs.

Chlamydia are obligate intracellular bacteria and important pathogens of humans and animals. Chlamydia-related bacteria are also major fish pathogens, infecting epithelial cells of the gills and skin to cause the disease epitheliocystis. Given the wide distribution, ancient origins and spectacular diversity of bony fishes, this group offers a rich resource for the identification and isolation of novel Chlamydia. The broad-nosed pipefish (Syngnathus typhle) is a widely distributed and genetically diverse temperate fish species, susceptible to epitheliocystis across much of its range. We describe here a new bacterial species, Candidatus Syngnamydia venezia; epitheliocystis agent of S. typhle and close relative to other chlamydial pathogens which are known to infect diverse hosts ranging from invertebrates to humans.