Laboratory mice with a wild microbiota generate strong allergic immune responses.

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early-life microbial exposures impede the development of subsequent allergic disease. Recently developed "wildling" mice are genetically identical to standard laboratory specific pathogen-free (SPF) mice but are housed under seminatural conditions and have rich microbial exposures from birth. Thus, by comparing conventional SPF mice with wildlings, we can uncouple the impact of lifelong microbial exposures from genetic factors on the allergic immune response. We found that wildlings developed larger populations of antigen-experienced T cells than conventional SPF mice, which included interleukin-10-producing CD4 T cells specific for commensal Lactobacilli strains and allergy-promoting T helper 2 (TH2) cells. In models of airway exposure to house dust mite (HDM), recombinant interleukin-33, or Alternaria alternata, wildlings developed strong allergic inflammation, characterized by eosinophil recruitment, goblet cell metaplasia, and antigen-specific immunoglobulin G1 (IgG1) and IgE responses. Wildlings developed robust de novo TH2 cell responses to incoming allergens, whereas preexisting TH2 cells could also be recruited into the allergic immune response in a cytokine-driven and TCR-independent fashion. Thus, wildling mice, which experience diverse and lifelong microbial exposures, were not protected from developing pathological allergic immune responses. Instead, wildlings mounted robust allergic responses to incoming allergens, shedding new light on the hygiene hypothesis.

Fluorescence enzymatic assay for bacterial polyphosphate kinase 1 (PPK1) as a platform for screening antivirulence molecules.

Inorganic polyphosphate (polyP) and its metabolic enzymes are important in several cellular processes related with virulence and antibiotic susceptibility. Accordingly, bacterial polyP synthesis has been proposed as a good target for designing novel antivirulence molecules as alternative to conventional antibiotics. In most pathogenic bacteria, polyphosphate kinase 1 (PPK1), in charge of polyP synthesis from ATP, is widely conserved. Current colorimetric and radioactive polyP synthesis enzymatic assays are not suitable for high-throughput screening of PPK1 inhibitors. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4'-6-diamidino-2-phenylindole), a fluorescence assay was previously developed by using a purified recombinant PPK1 enzyme from Escherichia coli. In this work we have developed a suitable methodology for high-throughput measurement of E. coli PPK1 activity. This platform can be used for the screening putative antimicrobial molecules for related enteropathogenic bacteria.

Dictyostelium discoideum as a surrogate host-microbe model for antivirulence screening in Pseudomonas aeruginosa PAO1.

The interest of the pharmaceutical industry in developing new antibiotics is decreasing, as established screening systems which identify compounds that kill or inhibit the growth of bacteria can no longer be used. Consequently, antimicrobial screening using classical minimum inhibitory concentration (MIC) measurements is becoming obsolete. The discovery of antimicrobial agents that specifically target a bacterial pathogen without affecting the host and its beneficial bacteria is a promising strategy. However, few host-microbe models are available for in vivo screening of novel antivirulence molecules. Here we designed high-throughput developmental assays in the social amoeba Dictyostelium discoideum to measure Pseudomonas aeruginosa virulence and to screen for novel antivirulence molecules without side effects to the host and its beneficial bacteria Klebsiella aerogenes. Thirty compounds were evaluated that had been previously selected by virtual screening for inhibitors of P. aeruginosa PAO1 polyphosphate kinase 1 (PaPPK1) and diverse compounds with combined PPK1 inhibitory and antivirulence activities were identified. This approach demonstrates that D. discoideum is a suitable surrogate host for preliminary high-throughput screening of antivirulence agents and that PPK1 is a suitable target for developing novel antivirulence compounds that can be further validated in mammalian models.