Significance: Novel antibiotics are urgently needed to resolve the current antimicrobial resistance crisis. For critical pathogens, drug entry through the cell envelope is one of the major challenges in the development of effective novel antibiotics. Envelope proteins forming water-filled channels, so-called porins, are commonly thought to be essential for entry of hydrophilic molecules, but we show here for the critical pathogen Pseudomonas aeruginosa that almost all antibiotics and diverse hydrophilic nutrients bypass porins and instead permeate directly through the outer membrane lipid bilayer. However, carboxylate groups hinder bilayer penetration, and Pseudomonas thus needs porins for efficient utilization of carboxylate-containing nutrients such as succinate. The major porin-independent entry route might open opportunities for facilitating drug delivery into bacteria.

Significance: Persisters represent a bacterial subpopulation that survive high antibiotic concentrations without being resistant. Their role in clinics in persistent infections and their molecular and functional landscape is not fully established. Staphylococcus aureus is a pathobiont that causes severe invasive infections often difficult to treat. Here, we assessed S. aureus recovered directly from persistent infections and show that host-mediated stress and antibiotic exposure promoted persister formation. Using a multiomics approach and enrichment of persisters, we were able to draw a molecular atlas of persisters, to correlate accumulation of insoluble proteins and ATP depletion with dormancy and persistence. Our results give insights into the molecular profile of bacterial persisters and provide a guide for therapy optimization for persistent S. aureus infections.

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase–, mitogen-activated protein kinase–, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.

The influenza pH1N1 virus caused a global flu pandemic in 2009 and continues manifestation as a seasonal virus. Better understanding of the virus-host cell interaction could result in development of better prevention and treatment options. Here we show that the Akt inhibitor MK2206 blocks influenza pH1N1 virus infection in vitro . In particular, at noncytotoxic concentrations, MK2206 alters Akt signaling and inhibits endocytic uptake of the virus. Interestingly, MK2206 is unable to inhibit H3N2, H7N9, and H5N1 viruses, indicating that pH1N1 evolved specific requirements for efficient infection. Thus, Akt signaling could be exploited further for development of better therapeutics against pH1N1 virus.