Correction: Effector-mediated subversion of proteasome activator (PA)28αß enhances host defense against Legionella pneumophila under inflammatory and oxidative stress conditions.

[This corrects the article DOI: 10.1371/journal.ppat.1011473.].

Effector-mediated subversion of proteasome activator (PA)28αß enhances host defense against Legionella pneumophila under inflammatory and oxidative stress conditions.

Legionella pneumophila is a natural pathogen of amoebae that causes Legionnaires' Disease in immunocompromised individuals via replication within macrophages. L. pneumophila virulence and intracellular replication hinges on hundreds of Dot/Icm-translocated effector proteins, which are essential for biogenesis of the replication-permissive Legionella-containing vacuole (LCV). However, effector activity can also enhance mammalian host defense via effector-triggered immunity. The L. pneumophila effector LegC4 is important for virulence in amoebae but enhances host defense against L. pneumophila in the mouse lung and, uniquely, within macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. The mechanism by which LegC4 potentiates cytokine-mediated host defense in macrophages is unknown. Here, we found that LegC4 enhances cytokine-mediated phagolysosomal fusion with Legionella-containing vacuole (LCV) and binds host proteasome activator (PA)28α, which forms a heterooligomer with PA28ß to facilitate ubiquitin-independent proteasomal degradation of oxidant-damaged (carbonylated) proteins. We found that oxidative stress was sustained in the presence of LegC4 and that the LegC4 restriction phenotype was relieved in PA28αß-deficient macrophages and in the lungs of mice in vivo. Our data also show that oxidative stress is sufficient for LegC4-mediated restriction in macrophages producing PA28αß. PA28αß has been traditionally associated with antigen presentation; however, our data support a novel mechanism whereby effector-mediated subversion of PA28αß enhances cell-autonomous host defense against L. pneumophila under inflammatory and oxidative stress conditions. This work provides a solid foundation to evaluate induced proteasome regulators as mediators of innate immunity.

Polymer Surface Dissection for Correlated Microscopic and Compositional Analysis of Bacterial Aggregates during Membrane Biofouling.

Multispecies biofilms are a common limitation in membrane bioreactors, causing membrane clogging, degradation, and failure. There is a poor understanding of biological fouling mechanisms in these systems due to the limited number of experimental techniques useful for probing microbial interactions at the membrane interface. Here, we develop a new experimental method, termed polymer surface dissection (PSD), to investigate multispecies assembly processes over membrane surfaces. The PSD method uses photodegradable polyethylene glycol hydrogels functionalized with bioaffinity ligands to bind and detach microscale, microbial aggregates from the membrane for microscopic observation. Subsequent exposure of the hydrogel to high resolution, patterned UV light allows for controlled release of any selected aggregate of desired size at high purity for DNA extraction. Follow-up 16S community analysis reveals aggregate composition, correlating microscopic images with the bacterial community structure. The optimized approach can isolate aggregates with microscale spatial precision and yields genomic DNA at sufficient quantity and quality for sequencing from aggregates with areas as low as 2000 µm2, without the need of culturing for sample enrichment. To demonstrate the value of the approach, PSD was used to reveal the composition of microscale aggregates of different sizes during early-stage biofouling of aerobic wastewater communities over PVDF membranes. Larger aggregates exhibited lower diversity of bacterial communities, and a shift in the community structure was found as aggregate size increased to areas between 25,000 and 45,000 µm2, below which aggregates were more enriched in Bacteroidetes and above which aggregates were more enriched with Proteobacteria. The findings demonstrate that community succession can be observed within microscale aggregates and that the PSD method is useful for identification and characterization of early colonizing bacteria that drive biofouling on membrane surfaces.