Pyruvate Oxidase as a Key Determinant of Pneumococcal Viability during Transcytosis across Brain Endothelium.

Streptococcus pneumoniae invades a myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defenses governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs), formed following S. pneumoniae internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which was previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar-metabolizing enzyme that catalyzes oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of an SpxB-catalyzed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. As expected, a ΔspxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defenses to restrict microbial passage into sterile compartments. Here, by focusing on the blood-brain barrier endothelium, we investigated mechanisms that enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar-metabolizing enzyme, was found to play a crucial role in this via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Direct ingestion, trophic transfer, and physiological effects of microplastics in the early life stages of Centropristis striata, a commercially and recreationally valuable fishery species.

Microplastics are ubiquitous in marine and estuarine ecosystems, and thus there is increasing concern regarding exposure and potential effects in commercial species. To address this knowledge gap, we investigated the effects of microplastics on larval and early juvenile life stages of the Black Sea Bass (Centropristis striata), a North American fishery. Larvae (13-14 days post hatch, dph) were exposed to 1.0 × 104, 1.0 × 105, and 1.0 × 106 particles L-1 of low-density polyethylene (LDPE) microspheres (10-20 µm) directly in seawater and via trophic transfer from microzooplankton prey (tintinnid ciliates, Favella spp.). We also compared the ingestion of virgin and chemically-treated microspheres incubated with either phenanthrene, a polycyclic aromatic hydrocarbon, or 2,4-di-tert-butylphenol (2,4-DTBP), a plastic additive. Larval fish did not discriminate between virgin or chemically-treated microspheres. However, larvae did ingest higher numbers of microspheres through ingestion of microzooplankton prey than directly from the seawater. Early juveniles (50-60 dph) were directly exposed to the virgin and chemically-treated LDPE microspheres, as well as virgin LDPE microfibers for 96 h to determine physiological effects (i.e., oxygen consumption and immune response). There was a significant positive relationship between oxygen consumption and increasing microfiber concentration, as well as a significant negative relationship between immune response and increasing virgin microsphere concentration. This first assessment of microplastic pollution effects in the early life stages of a commercial finfish species demonstrates that trophic transfer from microzooplankton can be a significant route of microplastic exposure to larval stages of C. striata, and that multi-day exposure to some microplastics in early juveniles can result in physiological stress.